CN116764694A

CN116764694A - Implementing barriers for controlled environments during sample processing and detection

Info

Publication number: CN116764694A
Application number: CN202310735022.1A
Authority: CN
Inventors: 内森·贝克特; 内森·卡斯韦尔
Original assignee: Altima Genomics
Current assignee: Altima Genomics
Priority date: 2018-12-07
Filing date: 2019-12-06
Publication date: 2023-09-19
Also published as: WO2020118172A1; KR20210118072A; US10512911B1; JP2022513737A; CN113490547A; CN113490547B; US20200179926A1; EP3890882A1; US11396015B2; CA3122370A1; IL296318A; US11648554B2; US20200179925A1; AU2019392781A1; IL283716B; AU2019392781B2; IL283716B2; EP3890882A4; IL296318B1; IL283716A

Abstract

Provided herein are methods for processing and/or detecting a sample. The method may include providing a barrier between a first region and a second region, wherein the first region contains the sample, wherein the barrier maintains the first region under a first atmosphere different from a second atmosphere of the second region, wherein a portion of the barrier comprises a coherently moving fluid; and detecting one or more signals from the sample using a detector at least partially contained in the first region while maintaining the first region at a first atmosphere different from a second atmosphere of the second region. The fluid-containing barrier portion may have a pressure that is lower than the first atmosphere, the second atmosphere, or both.

Description

Implementing barriers for controlled environments during sample processing and detection

The application is a divisional application of China patent application (corresponding to PCT application of which the application date is 2019, 12, 06 and PCT/US 2019/064916) with the application date of 2019, 12, 06 and the application number of 201980091436.7 and the application name of 'barrier realization of controlled environment during sample treatment and detection'.

Cross reference

The present application claims the benefit of U.S. patent application Ser. No. 16/665,559, U.S. patent application Ser. No. 16/665,540, U.S. patent application Ser. No. 16/440,026, and U.S. provisional patent application Ser. No. 62/776,866, each of which is incorporated herein by reference in its entirety, filed on Ser. No. 28, 10, 2019, 10, and filed on 13, 6, 2019.

Background

Biological sample processing has a variety of applications in the fields of molecular biology and medicine (e.g., diagnostics). For example, nucleic acid sequencing can provide information that can be used to diagnose a condition in a subject and, in some cases, to tailor a treatment plan. Sequencing is widely used in molecular biology applications including vector design, gene therapy, vaccine design, industrial strain design and validation. Biological sample processing may involve a fluidic system and/or a detection system.

Disclosure of Invention

Samples, including biological samples and non-biological samples, may be processed in a controlled environment, for example, with controlled temperature, pressure, and/or humidity. Analysis of such samples may involve detection of the sample within a controlled environment. Detection may involve continuous detection (e.g., continuous scanning) where there is continuous relative movement between the detector (e.g., optical head) and the sample. Detection may require proximity between the objective lens and the sample, for example to achieve direct or indirect contact between the objective lens and the sample. However, detection activities, such as the act of continuously scanning the sample, may disrupt the controlled environment. In some cases, efforts to maintain a controlled environment may disrupt the continuous motion of one or more detectors. In some cases, it may not be possible to move the detector within the controlled environment while maintaining the controlled environment, as, for example, the presence or movement of the detector may make sealing or maintaining the controlled environment difficult or impossible, or the presence or movement of the detector may affect the sample, thereby affecting the detection result. In some cases, mechanical seals, such as bellows or sliding gaskets, are implemented to keep the controlled environment separate from the normal environment (e.g., indoor environment), which may introduce undesirable forces during detection and impede or destroy the relative motion between the detector and the sample. Such problems may produce inaccurate and imprecise detection results. Accordingly, there is a recognized need for systems, devices, and methods that address at least the above-identified problems.

Provided herein are barriers that can be implemented between a controlled sample environment and an external environment. Such a barrier may allow low or zero friction relative motion between the detector and the sample while maintaining a controlled sample environment. The barrier may allow the objective lens to contact the sample directly or indirectly (e.g., by immersion in a fluid) during detection and movement. The barrier may allow for continuous scanning involving relative motion in a non-linear direction (e.g., in an R, θ coordinate system) and/or a linear direction (e.g., in X, Y and/or Z coordinate system). Advantageously, such barriers may allow for continuous scanning in a 100% or substantially 100% relative humidity environment. The barrier may prevent moisture from escaping the sample environment, which may condense and affect (e.g., corrode, contaminate, etc.) sensitive equipment, such as optics and electronics, when escaping. Furthermore, the barrier may prevent contaminants from the external environment from entering the sample environment, which may contaminate the sample and/or affect the fluid and/or detection (e.g., imaging).

The barrier may comprise a transition region between the sample environment and the external environment. The barrier may comprise a fluid barrier. The barrier may include fluid from the sample environment, the external environment, or both. The barrier may be a low pressure region. The low pressure region may have a lower pressure than the sample environment, the external environment, or both. The barrier may comprise a partial vacuum. The barrier may also include a physical barrier.

In one aspect, a method of processing a biological analyte is provided, comprising: (a) Providing a barrier between a first region and a second region, wherein the first region comprises a substrate having a biological analyte immobilized adjacent thereto, wherein the barrier maintains the first region under a first atmosphere different from a second atmosphere of the second region; and (b) detecting one or more signals from the biological analyte or a change thereof using a detector at least partially contained in the first region while (i) the detector is in motion relative to the substrate, wherein the substrate and the detector are not in direct mechanical contact, and (ii) the first region is maintained at a first atmosphere different from a second atmosphere of the second region.

In some embodiments, a portion of the barrier comprises a fluid that moves in its entirety. In some embodiments, the fluid comprises air. In some embodiments, the portion of the barrier comprises a partial vacuum. In some embodiments, the portion of the barrier comprises fluid from the first region, the second region, or both.

In some embodiments, the first atmosphere is maintained at a first humidity or first humidity range that is different from a second humidity or second humidity range of the second atmosphere. In some embodiments, the first atmosphere has a relative humidity of greater than 90%.

In some embodiments, the first atmosphere is maintained at a first temperature or first temperature range that is different from a second temperature or second temperature range of the second atmosphere.

In some embodiments, the first region comprises a first portion and a second portion, wherein the first portion is maintained under a first partial atmosphere, and wherein the second portion is maintained under a second partial atmosphere different from the first partial atmosphere. In some embodiments, the first local atmosphere is maintained at a first local temperature or first local temperature range that is different from a second local temperature or second local temperature range of the second local atmosphere. In some embodiments, the first local atmosphere is maintained at a first local humidity or first local humidity range that is different from a second local humidity or second local humidity range of the second local atmosphere.

In some embodiments, the detector is an optical detector and wherein the one or more signals are one or more optical signals or signal changes.

In some embodiments, the barrier comprises a first solid component and a second solid component, wherein the first solid component and the second solid component are not in direct mechanical contact, and wherein the first solid component is moveable relative to the second solid component. In some embodiments, a portion of the barrier comprises a bulk moving fluid, and wherein the portion is disposed between the first solid component and the second solid component.

In some embodiments, the detector is fixed relative to the first solid component and wherein the substrate is fixed (translationally fixed) in a translational direction relative to the second solid component.

In some embodiments, the substrate is rotatable relative to the second solid component.

In some embodiments, the first portion of the first solid component is disposed between the first region and the second region, and wherein the second portion of the first solid component is disposed between the second region and the third region to form a portion of another barrier configured to maintain the third region in a third atmosphere independent of the first atmosphere and the second atmosphere, wherein a portion of the other barrier comprises a fluid that moves in bulk, and wherein the third region is movable relative to the first solid component independent of the first region.

In some embodiments, the second atmosphere is a room atmosphere or an ambient atmosphere.

In some embodiments, the first portion of the detector is in a first region and the second portion of the detector is in a second region. In some embodiments, the first portion of the detector includes an optical imaging objective at least partially immersed in an immersion fluid in contact with the substrate in the first region.

In some embodiments, the biological analyte is a nucleic acid molecule, and further comprises identifying the sequence of the nucleic acid molecule or derivative thereof based at least in part on one or more signals or changes thereof.

In some embodiments, the movement comprises one or more selected from the group consisting of: (i) Substantially linear movement relative to the substrate and (ii) substantially non-linear movement.

In some embodiments, the detector performs a rotational motion relative to the substrate.

In some embodiments, the detector performs translational movement relative to the substrate.

In some embodiments, the detector performs translational and rotational movements relative to the substrate.

In some embodiments, in (b), the detector scans the substrate along a substantially linear scan path.

In some embodiments, in (b), the detector scans the substrate along a substantially non-linear scan path. In some embodiments, in (b), the detector scans the substrate along one or more scan paths selected from the group consisting of loops, spirals, and arcs.

In another aspect, a method for processing a biological analyte is provided, comprising: (a) Providing a barrier between the first region and the second region, wherein the first region contains a biological analyte, wherein the barrier maintains the first region under a first atmosphere different from a second atmosphere of the second region, wherein a portion of the barrier comprises a bulk moving fluid; and (b) detecting one or more signals from the biological analyte or a change thereof using a detector at least partially contained in the first region while the first region is maintained under a first atmosphere different from a second atmosphere of the second region.

In some embodiments, the portion of the barrier comprises fluid from the first region, the second region, or both.

In some embodiments, (b) comprises moving the detector relative to the biological analyte at the time of detection.

In some embodiments, the detector is an optical detector, and wherein the one or more signals or variations thereof are one or more optical signals or variations thereof.

In some embodiments, the barrier comprises a first solid component and a second solid component, wherein the first solid component and the second solid component are not in mechanical contact, and wherein the first solid component is movable relative to the second solid component. In some embodiments, the portion of the barrier comprising the fluid is disposed between the first solid component and the second solid component.

In some embodiments, the detector is immobilized with respect to the first solid component and wherein the biological analyte is immobilized in a translational direction with respect to the second solid component.

In some embodiments, a first portion of the first solid component is disposed between the first region and the second region, and wherein a second portion of the first solid component is disposed between the second region and the third region to form a portion of another barrier configured to maintain the third region under a third atmosphere independent of the first atmosphere and the second atmosphere, wherein a portion of the other barrier comprises a fluid, and wherein the third region is movable relative to the first solid component independent of the first region.

In some embodiments, the first portion of the detector is in a first region and the second portion of the detector is in a second region. In some embodiments, the first portion of the detector includes an optical imaging objective at least partially immersed in an immersion fluid in contact with the biological analyte in the first region.

In some embodiments, the biological analyte is a nucleic acid molecule, and further comprises identifying the sequence of the nucleic acid molecule or derivative thereof based at least in part on one or more signals or signal changes.

In some embodiments, the fluid comprises air.

In another aspect, a system for processing an analyte is provided, comprising: a first region configured to comprise (i) a substrate comprising an analyte immobilized adjacent thereto and (ii) at least a portion of a detector; and a barrier disposed between the first region and the second region, wherein the barrier is configured to maintain the first region under a first atmosphere different from a second atmosphere of the second region upon relative movement of the detector and the substrate with respect to each other to detect one or more signals from the analyte or changes thereof.

In some embodiments, a portion of the barrier is configured to include a fluid that moves in its entirety. In some embodiments, a portion of the barrier is configured to be under vacuum. In some embodiments, a portion of the barrier is configured to include fluid from the first region, the second region, or both the first region and the second region.

In some embodiments, a portion of the barrier is configured to include air.

In some embodiments, the barrier is configured to maintain the first region at a first humidity or first humidity range, wherein the first humidity or first humidity range is different from the second humidity or second humidity range of the second region. In some embodiments, the first atmosphere has a relative humidity of greater than 90%.

In some embodiments, the barrier is configured to maintain the first region at a first temperature or a first temperature range, wherein the first temperature or the first temperature range is different from a second temperature or a second temperature range of the second region.

In some embodiments, the first region includes a first portion and a second portion, wherein the barrier is configured to maintain the first portion under a first partial atmosphere and to maintain the second portion under a second partial atmosphere different from the first partial atmosphere. In some embodiments, the barrier is configured to maintain the first local atmosphere at a first local temperature or first local temperature range that is different from a second local temperature or second local temperature range of the second local atmosphere. In some embodiments, the barrier is configured to maintain the first local atmosphere at a first local humidity or first local humidity range different from a second local humidity or second local humidity range of the second local atmosphere.

In some embodiments, the detector is at least partially contained in the first region. In some embodiments, the detector is an optical detector and wherein the one or more signals are one or more optical signals or signal changes. In some embodiments, the first portion of the detector is in a first region and the second portion of the detector is in a second region. In some embodiments, the first portion of the detector includes an optical imaging objective configured to be at least partially immersed in an immersion fluid in contact with the substrate when the substrate is in the first region. In some embodiments, the detector is configured to move while the substrate is stationary. In some embodiments, the substrate is configured to move while the detector is stationary.

In some embodiments, the barrier comprises a first solid component and a second solid component, wherein the first solid component and the second solid component are not in direct mechanical contact with each other, and wherein the first solid component and the second solid component are movable relative to each other. In some embodiments, a portion of the barrier is configured to include the fluid in bulk motion, and wherein the portion is disposed between the first solid component and the second solid component.

In some embodiments, the detector is configured to be fixed relative to the first solid component, and wherein the substrate is configured to be fixed relative to the second solid component.

In some embodiments, the detector is configured to be fixed relative to the first solid component, and wherein the substrate is configured to be rotatable relative to the second solid component.

In some embodiments, the first portion of the first solid component is disposed between the first region and the second region, and wherein the second portion of the first solid component is disposed between the second region and the third region to form a portion of another barrier configured to maintain the third region under a third atmosphere independent of the first atmosphere and the second atmosphere, wherein a portion of the other barrier comprises a fluid that moves in bulk, and wherein the third region is movable relative to the first solid component independent of the first region.

In another aspect, a system for processing or analyzing an analyte is provided, comprising: a chamber and a lid, wherein the chamber comprises a first region configured to contain (1) a substrate containing an analyte immobilized thereabout, and (2) at least a portion of a detection unit, and wherein the lid is configured to be disposed adjacent to the chamber; and a fluid flow unit configured to provide fluid in an overall motion at a position disposed between the chamber and the cover when the cover is disposed adjacent to the chamber, such that the first region is maintained under a first atmosphere different from a second atmosphere of a second region located outside the first region.

In some embodiments, the bulk moving fluid is configured to provide a partial vacuum between the chamber and the lid.

In some embodiments, the fluid flow unit is configured to provide fluid for overall movement using fluid from the first region, the second region, or both.

In some embodiments, the fluid comprises air.

In some embodiments, wherein the fluid flow unit is configured to maintain the first region at a first humidity or first humidity range, wherein the first humidity or first humidity range is different from the second humidity or second humidity range of the second region. In some embodiments, the first atmosphere has a relative humidity of greater than 90%.

In some embodiments, the fluid flow unit is configured to maintain the first region at a first temperature or a first temperature range, wherein the first temperature or the first temperature range is different from a second temperature or a second temperature range of the second region.

In some embodiments, the first region comprises a first portion and a second portion, wherein the fluid flow unit is configured to maintain the first portion under a first partial atmosphere and to maintain the second portion under a second partial atmosphere different from the first partial atmosphere. In some embodiments, the fluid flow unit is configured to maintain the first local atmosphere at a first local temperature or first local temperature range different from a second local temperature or second local temperature range of the second local atmosphere. In some embodiments, the fluid flow unit is configured to maintain the first local atmosphere at a first local humidity or first local humidity range different from a second local humidity or second local humidity range of the second local atmosphere.

In some embodiments, the detection unit is at least partially contained in the first region. In some embodiments, the detection unit is an optical detection unit. In some embodiments, the first portion of the detection unit is in a first region and the second portion of the detection unit is in a second region. In some embodiments, the first portion of the detection unit includes an optical imaging objective configured to be at least partially immersed in an immersion fluid in contact with the substrate in the first region. In some embodiments, the detection unit is configured to move while the substrate is stationary. In some embodiments, the substrate is configured to move when the detection unit is stationary. In some embodiments, the relative motion comprises one or more selected from the group consisting of: (i) Substantially linear motion and (ii) substantially non-linear motion. In some embodiments, the detection unit is configured to be fixed relative to the cover. In some embodiments, the substrate is configured to be rotatable relative to the chamber.

In some embodiments, the detection unit includes one or more optics.

In some embodiments, the detection unit includes a sensor configured to capture a signal from the analyte.

In some embodiments, the chamber is not in mechanical contact with the lid.

In some embodiments, the lid is configured to move relative to the chamber, or vice versa.

In some embodiments, the fluid flow unit is configured to maintain the first region under the first atmosphere when the detection unit and the substrate are moved relative to each other.

In some embodiments, the fluid flow unit is configured to generate a negative pressure in a location disposed between the chamber and the lid.

In some embodiments, the first portion of the cover is disposed between the first region and the second region, and wherein the second portion of the cover is disposed between the second region and the third region, wherein the second fluid flow unit is configured to provide a fluid that moves in bulk to maintain the third region under a third atmosphere that is independent of the first atmosphere and the second atmosphere, and wherein the third region is movable relative to the cover independent of the first region.

In some embodiments, the system further comprises a controller operably coupled to the fluid flow unit, wherein the controller is configured to direct the fluid flow unit to cause the fluid to perform an overall motion.

In another aspect, a system is provided, comprising: an imaging objective configured to detect a signal or signal change from an analyte coupled to a substrate; a housing configured to hold a volume of fluid between the imaging objective and the substrate; a fluid source configured to comprise an aqueous solution; and a fluid flow unit configured to deliver the volume of fluid from the fluid source to the housing.

In some embodiments, wherein the aqueous solution comprises a wash solution.

In some embodiments, the aqueous solution comprises an immersion buffer solution comprising a salt, a surfactant, and a buffer.

In some embodiments, the aqueous solution has a pH of 8.0 to 9.0.

In some embodiments, the system further comprises a substrate. In some embodiments, the substrate comprises a fluid layer comprising the second aqueous solution. In some embodiments, the aqueous solution and the second aqueous solution comprise different compositions. In some embodiments, the aqueous solution and the second aqueous solution comprise the same composition.

In another aspect, a method is provided, comprising: (a) Contacting the imaging objective with a volume of fluid, wherein the fluid comprises a first aqueous solution, wherein the substrate comprises (i) an analyte immobilized thereabout, and (ii) a fluid layer adjacent thereto, wherein the fluid layer comprises a second aqueous solution; and (b) imaging the analyte through the volume of fluid by an imaging objective.

In some embodiments, the method further comprises moving the imaging objective relative to the substrate while maintaining fluid contact between the imaging objective and the substrate.

In some embodiments, the method further comprises moving the substrate relative to the imaging objective while maintaining fluid contact between the imaging objective and the substrate.

In some embodiments, the volume of fluid has a thickness of about 200 micrometers (μm) to 500 μm.

In some embodiments, the fluid layer has a thickness of about 5 μm to 50 μm.

In some embodiments, the method further comprises (i) breaking fluid contact between the imaging objective and the substrate, and (ii) bringing the imaging objective and the substrate into a second fluid contact. In some embodiments, after (i), at least a portion of the volume of fluid remains in fluid contact with the imaging objective. In some embodiments, after (i), at least a portion of the volume of fluid remains in contact with the substrate fluid.

In some embodiments, the first aqueous solution comprises a wash solution.

In some embodiments, the first aqueous solution comprises an immersion buffer solution comprising a salt, a surfactant, and a buffer.

In some embodiments, the first aqueous solution has a pH of 8.0 to 9.0.

In some embodiments, the first aqueous solution and the second aqueous solution comprise different compositions.

In some embodiments, the first aqueous solution and the second aqueous solution comprise the same composition.

In another aspect, a method is provided, comprising: (a) Contacting the imaging objective with an analyte fluid immobilized adjacent to a substrate by a volume of fluid, wherein the substrate comprises a fluid layer comprising a second aqueous solution; and (b) imaging the analyte through the volume of fluid by an imaging objective.

In some embodiments, the method further comprises moving the imaging objective relative to the analyte while maintaining fluid contact between the imaging objective and the analyte.

In some embodiments, the method further comprises moving the analyte relative to the imaging objective while maintaining fluid contact between the imaging objective and the analyte.

In some embodiments, the volume of fluid has a thickness of about 200 μm to 500 μm.

In some embodiments, the fluid layer has a thickness of about 5 μm to 50 μm.

In some embodiments, the method further comprises (i) breaking fluid contact between the imaging objective and the analyte, and (ii) bringing the imaging objective and the analyte into a second fluid contact. In some embodiments, after (i), at least a portion of the volume of fluid remains in fluid contact with the imaging objective. In some embodiments, after (i), at least a portion of the volume of fluid remains in contact with the analyte fluid.

In some embodiments, the first aqueous solution comprises a wash solution.

In some embodiments, the first aqueous solution has a pH of 8.0 to 9.0.

In another aspect, a system for processing or analyzing an analyte is provided, comprising: a chamber and a lid, wherein the chamber includes an interior region and is configured to include a substrate configured to immobilize the analyte in proximity thereto, wherein the lid is configured to be disposed adjacent to the chamber; an environmental unit configured to maintain a first local environment, a second local environment, and a third local environment within the interior region, wherein the environmental unit is configured to maintain (i) the first local environment at a first temperature or temperature range, (ii) the second local environment at a second temperature or temperature range, and (iii) the third local environment at a third temperature or temperature range, wherein the first local environment is disposed above the second local environment and the third local environment, and wherein the first local environment is located at or near the lid, and wherein the second local environment is disposed at or near the surface of the substrate, and wherein the third local environment is disposed below the first local environment and the second local environment, and wherein the first temperature or temperature range is higher than the second temperature or temperature range and the third temperature or temperature range, and wherein the second temperature or temperature range is lower than the third temperature or temperature range.

Other aspects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.

Incorporation by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in this specification, this specification is intended to supercede and/or take precedence over any such contradictory material.

Drawings

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "figures") in which

In the figure:

fig. 1A illustrates a cross-sectional side view of an example barrier system.

Fig. 1B shows a perspective view of fig. 1A.

Fig. 1C shows a cross-sectional view of an exemplary immersion optical system.

Fig. 2A shows a partial cross-sectional view of a barrier system holding a fluid barrier.

Fig. 2B shows a reduced view of the barrier system of fig. 2A.

Fig. 2C shows a perspective view of a chamber of the barrier system of fig. 2A.

Figure 3 shows a barrier system with multiple sample environments.

Fig. 4 illustrates an exemplary barrier system including different local environments.

Fig. 5 illustrates a processing system including an exemplary barrier system.

Fig. 6 shows an example of an array on a substrate.

FIG. 7 illustrates a computer control system programmed or otherwise configured to implement the methods provided herein;

fig. 8 shows an example of an image generated by imaging a substrate immobilized with a biological analyte in a sample environment of a barrier system of the present disclosure.

Fig. 9 shows signal data processed by imaging a substrate immobilized with a biological analyte in a sample environment of a barrier system of the present disclosure.

Detailed Description

While various embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

Provided herein are barriers that can be implemented between a controlled sample environment and an external environment. The barrier may comprise a transition region between the sample environment and the external environment. The barrier may comprise a fluid barrier. The barrier may include fluid from the sample environment, the external environment, or both. The barrier may be a low pressure region. The low pressure region may have a lower pressure than the sample environment, the external environment, or both. The barrier may comprise a partial vacuum. The barrier may also include a physical barrier.

Advantageously, such a barrier may allow zero or low friction relative movement between the detector and the sample while maintaining a controlled sample environment. The barrier may allow for continuous scanning involving relative motion in a non-linear direction (e.g., in an R, θ coordinate system) and/or a linear direction (e.g., in X, Y and/or Z coordinate system). The barrier may allow for continuous scanning in a 100% or substantially 100% relative humidity environment. The barrier may prevent moisture from escaping the sample environment, which may condense and affect (e.g., corrode, contaminate, etc.) sensitive equipment, such as optics, when escaping. Furthermore, the barrier may prevent contaminants from the external environment from entering the sample environment, which may affect the fluid and/or detection (e.g., imaging).

As used herein, the term "fluid" generally refers to a gas or a liquid, or a mixture thereof. The fluid may include solid particles, liquid particles (e.g., water droplets), gas particles (e.g., inert gas atoms or non-inert gas molecules), or mixtures thereof. The fluid may comprise steam. The fluid may comprise a moisture content. The fluid may include air, such as ambient air, room air, atmospheric air, and/or pressurized air. The fluid may comprise a concentrated element or compound, either in isolated or mixed form, for example at a concentration of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% in the mixture. Alternatively or additionally, the fluid may comprise concentrated elements or compounds in a concentration of up to about 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in the mixture. The fluid may comprise a suspension or mixture of any particles in a gaseous medium. The fluid may comprise a suspension or mixture of any particles in a liquid medium. The fluid may comprise a mist, fog, vapor or aerosol. In some cases, the fluid may comprise a plasma. The volume of fluid may be capable of flowing, for example in the form of random, coherent, and/or global motion. The volume of fluid may have a net average motion directed in one or more directions or toward a reference destination. In some cases, a volume of fluid moving coherently or integrally may have streamlines oriented in the same general direction. A volume of fluid that is in coherent or global motion may be distinguished from fluid that is in random motion (e.g., not in coherent motion, not in global motion, not having a net average motion). The volume of fluid may have turbulent and/or laminar flow.

As used herein, the term "sample" generally refers to a biological sample. The systems, devices, and methods provided herein may be particularly advantageous for analyzing biological samples that may be highly sensitive to the environment, for example, to the temperature, pressure, and/or humidity of the environment. The biological sample may be from any subject or living organism. For example, the subject may be an animal, mammal, bird, vertebrate, rodent (e.g., mouse), primate, simian, human, or other organism, e.g., a plant. Animals may include, but are not limited to, farm animals, sports animals, and pets. The subject may be a healthy or asymptomatic individual, an individual who has or is suspected of having a disease (e.g., cancer) or is susceptible to the disease, and/or an individual in need of treatment or suspected of requiring treatment. The subject may be a patient. The subject may be a microorganism (microorgan) or a microorganism (microbe) (e.g., bacteria, fungi, archaea, viruses).

The biological sample may comprise any number of macromolecules, such as cellular macromolecules. The biological sample may be a cell sample. The biological sample may be a cell line or a cell culture sample. The biological sample may include one or more cells. The biological sample may include one or more microorganisms. The biological sample may be a nucleic acid sample or a protein sample. The biological sample may also be a carbohydrate sample or a lipid sample. The biological sample may be from another sample. The sample may be a tissue sample, such as a biopsy, a nuclear biopsy, a needle aspirate, or a fine needle aspirate. The sample may be a fluid sample, such as a blood sample, a urine sample, or a saliva sample. The sample may be a skin sample. The sample may be a cheek swab. The sample may be a plasma or serum sample. The sample may be cell-free or a cell-free sample. The cell-free sample may comprise extracellular polynucleotides. The extracellular polynucleotides may be isolated from a body sample selected from the group consisting of blood, plasma, serum, urine, saliva, mucosal excretions, sputum, stool, and tears.

The biological sample may comprise one or more biological particles. The biological particles may be macromolecules. The biological particles may be small molecules. The biological particle may be a virus. The biological particles may be cells or cell derivatives. The biological particles may be organelles. The biological particles may be rare cells from a population of cells. The biological particles may be any type of cell including, but not limited to, prokaryotic cells, eukaryotic cells, bacteria, fungi, plant, mammalian or other animal cell types, mycoplasma, normal tissue cells, tumor cells, or any other cell type, whether derived from a single-cell or multicellular organism. The biological particle may be a component of a cell (e.g., a macromolecular component), such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), a nucleus, an organelle, a protein, a peptide, a polypeptide, or any combination thereof. The RNA may be encoded or non-encoded. For example, the RNA can be messenger RNA (mRNA), ribosomal RNA (rRNA), or transfer RNA (tRNA). The RNA may be a transcript. The RNA may be a small RNA less than 200 nucleobases in length, or a large RNA greater than 200 nucleobases in length. The micrornas may include 5.8S ribosomal RNAs (rrnas), 5S rrnas, transfer RNAs (trnas), micrornas (mirnas), small interfering RNAs (sirnas), micronucleolar RNAs (snoRNAs), piwi-interacting RNAs (pirnas), tRNA-derived micrornas (tsrnas), and small rDNA-derived RNAs (srrnas). The RNA may be double-stranded RNA or single-stranded RNA. The RNA may be circular RNA. The biological particles may be hardened cells. Such hardened cells may or may not include cell walls or cell membranes. Alternatively or additionally, the sample of the present disclosure may comprise a non-biological sample.

As used herein, the term "analyte" generally refers to a subject whose one or more characteristics being analyzed, measured, determined, or otherwise examined. The analyte may be a biological analyte, i.e. e.g. a biological sample, or derived from a biological sample. The analyte may be a non-biological analyte, i.e. for example a non-biological sample, or derived from a non-biological sample.

As used herein, the term "move relative to … …" or similar variants ("movable relative to … …" ) when referring to a relationship between a first object and a second object (e.g., movement of the first object relative to the second object) generally refers to movement of the first object, the second object, or both relative to the other.

As used herein, the term "detector" may refer to any device or component of a device configured to detect a signal. The detector may comprise an objective lens. The detector may comprise a plurality of objective lenses. The detector may comprise an imaging system.

Whenever the term "at least", "greater than" or "greater than or equal to" precedes the first value in a series of two or more values, the term "at least", "greater than" or "greater than or equal to" applies to each value in the series. For example, 1, 2, or 3 or more corresponds to 1 or more, 2 or 3 or more.

Whenever the term "no greater than", "less than" or "less than or equal to" precedes the first value in a series of two or more values, the term "no greater than", "less than" or "less than or equal to" applies to each value in the series of values. For example, less than or equal to 3, 2, or 1 corresponds to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

Fluid barrier

Provided herein are methods for processing and/or detecting a sample. In some cases, the method may include providing a barrier between a first region (e.g., a region containing the sample) and a second region (e.g., an outer region). The barrier may maintain the first region under a first atmosphere different from a second atmosphere of the second region. A portion of the barrier may include fluid that moves coherently or integrally. The first region may comprise a sample. Then, a detector at least partially contained in the first region may detect one or more signals from the sample while the first region is maintained at a first atmosphere different from a second atmosphere of the second region. The detector may not be in direct mechanical contact with the substrate contained in the first region. The substrate may comprise a sample thereon. The detector may be in fluid (or fluid) contact with the substrate (e.g., the detector may not be in direct mechanical contact with the substrate, but may be fluidly coupled to the substrate). The detector may be in contact with the substrate liquid. The detector may be in contact with the substrate gas.

In some cases, the method may include providing a barrier between a first region (e.g., a region containing the sample) and a second region (e.g., an outer region), wherein the barrier maintains the first region at a first atmosphere different from a second atmosphere of the second region. The first region may comprise a sample. The detector at least partially contained in the first region may then detect one or more signals from the sample while (i) the detector is performing a detection event, such as when (1) the detector is performing continuous low or zero friction movement relative to the first region, (2) the detector is performing discrete movement relative to the first region at different time intervals (e.g., in a discontinuous manner), and (ii) the first region is maintained at a first atmosphere different from a second atmosphere of the second region. The detection event may include imaging or scanning during relative movement between the detector and the sample. Detecting the event may include imaging or scanning while the detector and sample are stationary relative to each other. The detector may not be in direct mechanical contact with the substrate contained in the first region, wherein the substrate includes the sample thereon. The detector may be in fluid contact with the substrate. The detector may be in contact with the substrate liquid. The detector may be in contact with the substrate gas.

Provided herein are systems for processing and/or detecting samples. In some cases, the system may include a barrier disposed between a first region (e.g., a region containing the sample) and a second region (e.g., an outer region). The first region may be configured to contain a sample. The barrier may be configured to maintain the first region under a first atmosphere different from a second atmosphere of the second region. A portion of the barrier may include fluid that moves coherently or integrally. The system may include a detector at least partially contained in the first region. The detector may be configured to detect one or more signals from the sample while maintaining the first region at a first atmosphere different from a second atmosphere of the second region. In some cases, the detector may be configured to detect one or more signals from the sample while the detector is performing a detection event. For example, the detection event may include a continuous low friction or zero friction movement of the detector relative to the first region. For example, the detection event may include discrete movements of the detector relative to the first region at different time intervals (e.g., in a discontinuous manner). The detection event may include imaging or scanning during relative movement between the detector and the sample. Detecting the event may include imaging or scanning while the detector and sample are stationary relative to each other. In some cases, the first region may include a substrate comprising a sample thereon. For example, the sample may be fixed adjacent to the substrate. In some cases, the detector may not be in direct mechanical contact with the substrate. In some cases, the detector may be in fluid contact with the substrate. The detector may be in contact with the substrate liquid. The detector may be in contact with the substrate gas.

Fig. 1A and 1B illustrate an exemplary barrier system 100, showing a cross-sectional side view and a perspective view, respectively. A fluid barrier 113 may be implemented between the sample environment 105 (e.g., a first region) and the external environment 107 (e.g., a second region). The sample environment 105 may be a controlled environment that includes one or more samples. The external environment 107 may be a closed or open environment. In some cases, the external environment 107 may be an indoor environment or an ambient environment. In some cases, the external environment 107 may also be a controlled environment.

The sample environment 105 region may be defined by the chamber 115, the plate 103, and the fluid barrier 113. Fluid barrier 113 may be maintained between chamber 115 and the physical gap between plates 103. As used herein, the term "plate" is interchangeably referred to herein as a cover. In some cases, the physical gap may be large enough to allow fluid communication between the sample environment 105 and the external environment 107 when the fluid barrier 113 is otherwise not in place. The chamber 115 and the plate 103 may be independent such that the chamber 115 and the sample environment 105 region defined thereby may be movable relative to the plate 103. For example, the sample environment 105 region may be defined by different portions of the plate 103, with the chamber 115 being positioned differently relative to the plate 103. The relative motion between the chamber 115 and the plate 103 may be in any direction, such as in a non-linear direction (e.g., in an R, θ coordinate system) and/or a linear direction (e.g., in X, Y, and/or Z coordinate system). For example, the relative motion may be rotational about a central axis, or linear along any linear axis. In some cases, actuator units (e.g., linear platforms, motors, etc.) and/or structural units (e.g., beams, supports, rails, etc.) may limit relative movement between the chamber 115 and the plate 103.

The plate 103 and the chamber 115 may not be in direct mechanical contact such that there is a minimum distance between the plate and the chamber. The minimum distance between the plate 103 and the chamber 115 may be at least about 100 micrometers (μm), 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1 millimeter (mm), 2mm, 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, 9mm, 1 centimeter (cm), or more. Alternatively or additionally, the minimum distance may be at most about 1cm, 9mm, 8mm, 7mm, 6mm, 5mm, 4mm, 3mm, 2mm, 1mm, 950 μm, 900 μm, 850 μm, 800 μm, 750 μm, 700 μm, 650 μm, 600 μm, 550 μm, 500 μm, 450 μm, 400 μm, 350 μm, 300 μm, 250 μm, 200 μm, 150 μm, 100 μm or less. Alternatively or additionally, the minimum distance may be within a range defined by any two of the foregoing values.

The fluid barrier 113 may act as a transition region between the sample environment 105 and the external environment 107. The fluid barrier 113 may include a fluid (e.g., air) from the sample environment, the external environment, or both. The fluid barrier 113 may be a low pressure region. The fluid barrier 113 may have a lower pressure than the sample environment, the external environment, or both. The barrier may comprise a partial vacuum. The barrier may include one or more volumes of fluid subjected to negative pressure. In some cases, the fluid barrier 113 may be a high pressure region. For example, the fluid barrier may have a higher pressure than the sample environment, the external environment, or both. The fluid barrier 113 may be in coherent motion, such as in a coherent flow direction. The fluid barrier 113 may be in a unitary motion. The fluid barrier may include a volume of fluid having a net average motion oriented in one or more directions or toward a reference destination. In some cases, a volume of fluid in coherent or bulk motion may have streamlines oriented in the same general direction. Fluids that are coherently or integrally moved can be distinguished from fluids that are not part of the fluid barrier that are randomly moved (e.g., are not coherently moved, are not integrally moved, have no net average movement). The fluid in the fluid barrier may have turbulent and/or laminar flow.

The sample environment 105 may include a substrate. One or more samples may be immobilized on or adjacent to a substrate. Alternatively or additionally, one or more samples may be otherwise disposed on the substrate. In some cases, at least a portion of the chamber 115 may be or include a substrate. In other cases, the chamber 115 may be coupled to a substrate. In some cases, the substrate may be fixed relative to the chamber 115. Alternatively, the substrate may be movable relative to the chamber 115, such as in a linear and/or non-linear (e.g., rotational) direction. For example, the substrate may be fixed to the chamber 115 in XY coordinates (and/or Z coordinates), but rotatable relative to the chamber 115. Where the chamber 115 is movable relative to the plate 103 and the substrate is movable relative to the chamber 115, the two relative movements may or may not be operated by the same actuator unit.

The detector 101 may protrude from the external environment 107 into the sample environment 105 through the plate 103 (e.g., through a hole in the plate 103). The fit between the detector 101 and the aperture may be fluid tight such that there is no fluid communication through the aperture when the detector 101 is fitted through the aperture. Alternatively or additionally, the aperture may be hermetically sealed. Alternatively, the plate 103 may be integrated with the detector 101. Alternatively, the detector 101 may be entirely contained within the sample environment 105, for example, by securing the non-sample-facing end to the plate 103.

At least a portion of detector 101 may be fixed relative to plate 103. In some cases, the detector 101 may be capable of translating independently of the plate 103 along an axis substantially perpendicular to the plane of the plate 103 (e.g., through the aperture). In some cases, at least a portion of the detector 101 (e.g., a portion of the detector within the sample environment area) may be capable of moving (e.g., linearly or non-linearly, e.g., rotating) independent of the plate 103.

Within the sample environment 105, the detector 101 may be configured to detect one or more samples disposed on a substrate using an immersion optical system. A portion of the detector within the sample environment 105, such as an optical imaging objective, may be in optical communication with the substrate through the liquid fluid 131 medium. In some cases, the liquid fluidic medium may be disposed on a localized area of the substrate. In other cases, the liquid fluidic medium may be disposed over the entire area of the substrate surface (e.g., through the bottom of the chamber 115). Alternatively, the detector may be in optical communication with the substrate in the absence of a liquid fluid medium.

Fig. 1C shows a cross-sectional view of an exemplary immersion optical system 1100. The system 1100 may be used to optically image a substrate as described herein. The system 1100 may be integrated with any barrier system described elsewhere herein. The system may include an optical imaging objective 1110 (e.g., detector 101). For example, the objective may have projected into the sample environment (e.g., through plate 103) or may be contained in the sample environment (e.g., and secured to the surface of plate 103). The optical imaging objective may be an immersion optical imaging objective. The optical imaging objective may be configured in optical communication with the substrate 1160. The optical imaging objective may be partially or completely enclosed by the housing 1120. The housing may partially or completely surround the sample-facing end of the optical imaging objective. The housing may be secured to the optical imaging objective and/or the plate. The housing may have a generally cup-like shape or form. The housing may be any container. The housing may be configured to hold a fluid 1140 (e.g., water or an aqueous solution or oil or an organic solution) into which the optical imaging objective is to be immersed. The fluid may be in contact with the substrate 1160. Thus, the objective lens and the substrate may be in fluid contact, e.g. liquid contact.

In some cases, the objective lens and the substrate can be maintained in fluid contact (e.g., liquid contact) by the fluid 1140 as the objective lens 1110 and the substrate 1160 undergo relative motion (e.g., linear motion, non-linear motion, rotational motion, etc.) in a substantially X-Y plane.

In some cases, when the objective 1110 and the substrate 1160 are relatively moved along the Z-axis or another axis (having a Z-component), such as when the objective is out of fluid contact (e.g., liquid contact) with the substrate, for example, between scans of different rounds, at least a portion of a volume of fluid 1140 from previous fluid contact between the objective and the substrate may remain in contact with the objective. In some cases, at least a portion of the volume of fluid 1140 from previous fluid contact between the objective lens and the substrate may remain on the substrate. In some cases, such fluids may become part of an aqueous interface or environment of one or more layers of an aqueous solution or mixture disposed adjacent to a substrate. On the next fluid contact between the objective lens and the substrate, a new volume of fluid may be provided in the housing 1120, for example with the aid of a fluid flow tube 1130 described elsewhere herein.

The housing 1120 may be configured to maintain a minimum distance 1150 between the substrate and the housing to avoid contact between the housing and the substrate 1160 during movement of the substrate relative to the plate. The minimum distance may be at least about 100 nanometers (nm), 200nm, 300nm, 400nm, 500nm, 1 micrometer (μm), 2 μm, 3 μm, 4 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 1 millimeter (mm), or more. Alternatively or additionally, the minimum distance may be at most about 1mm, 500 μm, 400 μm, 300 μm, 200 μm, 100 μm, 50 μm, 40 μm, 30 μm, 20 μm, 10 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm, 500nm, 400nm, 300nm, 200nm, 100nm or less. Alternatively or additionally, the minimum distance may be within a range defined by any two of the foregoing values. Even with a minimum distance, the housing may contain fluid due to surface tension effects. The system may include a fluid flow tube 1130 configured to deliver a fluid 1140 to an interior of the enclosure. The volume of fluid 1140 in the housing may be replenished and/or washed continuously or intermittently via a fluid flow tube during a detection event. Advantageously, any contaminants in the fluid volume may be washed away during such replenishment and/or washing. For example, in some cases, a volume of fluid may be continuously replenished. During replenishment, a new volume of fluid may be directed to the housing, and at least a portion of the existing volume of fluid in the housing may wet onto the surface of the substrate, and in some cases, wet off the edges of the substrate surface. The existing fluid volume may become part of the aqueous interface or fluid layer of the substrate. The fluid flow tube may be connected to the housing by an adapter 1135. The adapter may comprise a threaded adapter, a compression adapter, or any other adapter.

The electric field application unit (not shown) may be configured to adjust the hydrophobicity of one or more surfaces of the container to retain at least a portion of the fluid in contact with the immersion objective and the open substrate, such as by applying an electric field.

Fluid 1140 may comprise water or an aqueous solution. The fluid may comprise an oil or an organic solution. The fluid may comprise a mixture of aqueous and non-aqueous solutions. In cases where the fluid comprises water or one or more aqueous solutions, such fluids may be advantageously particularly suitable for maintaining continuity of the aqueous interface or environment adjacent the substrate 1160 and facilitating interaction between the objective lens and the substrate. For example, the substrate 1160 may include one or more layers of an aqueous solution or mixture adjacent thereto, such as for use in chemical processing operations and/or to retain an analyte or sample disposed on the substrate. The volume(s) of fluid 1140 that is in contact with and/or moves relative to the one or more layers of aqueous solution or mixture adjacent the substrate may not destroy or minimize or mitigate destruction of such one or more layers of aqueous solution or mixture adjacent the substrate. For example, if a volume of fluid 1140 is not miscible with the composition of one or more layers adjacent to the substrate, the optical path from the objective lens to the sample on the substrate may be disrupted, the relative motion between the objective lens and the substrate may be disrupted, the aqueous interface or environment adjacent to the substrate may be disrupted, undesired residues may be generated or left in one or more layers adjacent to the substrate or the fluid volume or both, and/or any combination of the above may occur. In some cases, the fluid and the one or more layers of aqueous solution or mixture may comprise the same aqueous solution or mixture. In some cases, the fluid and the one or more layers of aqueous solution or mixture may comprise different aqueous solutions or mixtures.

In some examples, fluid 1140 includes an immersion buffer solution. The immersion buffer solution may have the same composition as the wash solution used during the chemical operation. The immersion buffer solution and/or the wash solution may comprise a combination of buffers, salts and surfactants. The buffer solution may have a pH of about 8.0 to 9.0. In some cases, the fluid may have a pH of at least about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, or greater. Alternatively or additionally, the fluid may have a pH of up to about 9.5, 9.4, 9.3, 9.2, 9.1, 9.0, 8.9, 8.8, 8.7, 8.6, 8.5, 8.4, 8.3, 8.2, 8.1, 8.0, 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5 or less. In some examples, the immersion buffer solution comprises 20 millimoles (mM) of tris (hydroxymethyl) aminomethane, 110mM of NaCl, and 0.1% of Triton-X100.

In some cases, the optical imaging objective 1110 and the housing 1120 may provide a physical barrier between a first location on the substrate where the chemical processing operation is performed and a second location on the substrate where the detection operation is performed. In this way, the chemical treatment operation and the detection operation can be performed under independent operation conditions, and contamination of the detector can be avoided. The first location and the second location may have different humidity, temperature, pressure or atmosphere mixtures.

A method of detecting one or more signals from an analyte or a change thereof may comprise using an immersion optical system. The method may include bringing the optical imaging objective 1110 into fluid contact with a substrate 1160 by providing a fluid 1140 in a housing 1120 between the objective and the substrate, the substrate 1160 including an analyte disposed thereon. The fluid flow tube 1130 may be used to continuously or intermittently replenish or wash fluid. The method may further comprise, prior to contacting the objective lens with the substrate fluid, cleaning the surface of the substrate. The method may further comprise, prior to cleaning the substrate surface, contacting the reaction mixture with the substrate surface to perform one or more chemical treatment operations. The washing operation may prevent contamination between one or more chemical treatment operations (e.g., by the reaction mixture) and the detection operation. For example, such washing operations may prevent nucleotides or other reagents from being carried over from chemical processing operations to imaging or scanning operations.

The method may further comprise removing the fluid contact between the objective lens and the substrate, for example by lifting the objective lens relative to the substrate and/or lowering the substrate relative to the objective lens.

The method further includes repeating the detecting operation multiple times on the same substrate (e.g., bringing the optical imaging objective into fluid contact with the substrate and removing the fluid contact). For example, the detection operation may be repeated at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more times on the same substrate.

Fluid 1140 may comprise water or an aqueous solution or mixture. The fluid may comprise an oil, a non-aqueous solution, and/or an organic solution or mixture. The substrate may include one or more layers of an aqueous solution or mixture adjacent thereto. The substrate may comprise one or more layers of oil, non-aqueous solutions, and/or organic solutions or mixtures adjacent thereto. Fluid 1140 and the layer or layers of solution or mixture adjacent thereto may each comprise a miscible composition.

Systems and methods for detection may include an immersion objective system as described herein.

The system may include an imaging objective configured to detect a signal or signal change from an analyte coupled to the substrate; a housing configured to hold a volume of fluid between the imaging objective and the substrate; a fluid source comprising an aqueous solution; and a fluid flow unit configured to deliver a volume of fluid from the fluid source to the housing. The housing may be a physical housing. The aqueous solution may include an immersion buffer solution. The substrate may include one or more fluid layers. The one or more fluid layers may comprise a second aqueous solution. The second aqueous solution and the aqueous solution may comprise different compositions (e.g., different salts or concentrations thereof, different surfactants or concentrations thereof, different buffers or concentrations thereof, different compounds or mixtures thereof, or concentrations thereof). The second aqueous solution and the aqueous solution may comprise the same solution. The analyte and the objective lens may be in fluid contact by the aqueous solution and the second aqueous solution. The system may also include a substrate. The substrate may be any substrate as described elsewhere herein.

A method is provided that includes contacting an imaging objective with a substrate fluid through a volume of fluid. The volume of fluid may comprise a first aqueous solution. The substrate may comprise (i) an analyte immobilized adjacent thereto, and (ii) a fluid layer adjacent thereto. The fluid layer may include a second aqueous solution. The method may include imaging the analyte through an imaging objective through a volume of fluid. The method may further comprise moving the imaging objective relative to the substrate or moving the substrate relative to the imaging objective, or both, while maintaining fluid contact between the imaging objective and the substrate. The method may further include (i) breaking fluid contact between the imaging objective and the substrate, and (ii) bringing the imaging objective and the substrate into fluid contact a second time. At least a portion of the volume of fluid may remain in fluid contact with the imaging objective and/or the substrate after the fluid contact is broken.

A method is provided that includes contacting an imaging objective with an analyte fluid immobilized adjacent to a substrate by a volume of fluid comprising a first aqueous solution. The substrate may include a layer of fluid that includes a second volume of fluid. The method may include imaging the analyte through an imaging objective through a volume of fluid. The method may further comprise moving the imaging objective relative to the substrate or moving the substrate relative to the imaging objective, or both, while maintaining fluid contact between the imaging objective and the substrate. The method may further include (i) breaking fluid contact between the imaging objective and the analyte, and (ii) bringing the imaging objective and the analyte into fluid contact a second time. At least a portion of the volume of fluid may remain in contact with the imaging objective and/or the analyte fluid after the fluid contact is broken.

The volume of fluid may have a thickness (e.g., minimum distance between the objective lens and the substrate and/or analyte) on the order of 10 μm, 100 μm, 1000 μm (or 1 millimeter (mm)), 10mm, 100mm, or more. In some cases, the fluid volume may be at least about 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, 260 μm, 270 μm, 280 μm, 290 μm, 300 μm, 310 μm, 320 μm, 330 μm, 340 μm, 350 μm, 360 μm, 370 μm, 380 μm, 390 μm, 400 μm, 410 μm, 420 μm, 430 μm, 440 μm, 450 μm, 460 μm, 470 μm, 480 μm, 490 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1000 μm or more thick. Alternatively or additionally, the thickness of the volume of fluid may have a thickness of at most about 1000 μm, 950 μm, 900 μm, 850 μm, 800 μm, 750 μm, 700 μm, 650 μm, 600 μm, 550 μm, 500 μm, 490 μm, 480 μm, 470 μm, 460 μm, 450 μm, 440 μm, 430 μm, 420 μm, 410 μm, 400 μm, 390 μm, 380 μm, 370 μm, 360 μm, 350 μm, 340 μm, 330 μm, 320 μm, 310 μm, 300 μm, 290 μm, 280 μm, 270 μm, 260 μm, 250 μm, 240 μm, 230 μm, 220 μm, 210 μm, 200 μm, 190 μm, 180 μm, 170 μm, 160 μm, 150 μm, 140 μm, 130 μm, 120 μm, 110 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm or less. Alternatively or additionally, the thickness of the volume of fluid may be between any range of any two of the foregoing values. The substrate may include one or more fluid layers, each layer having the same or different fluid composition. The fluid layer may comprise an aqueous solution. The fluid layer may comprise a non-aqueous solution. The fluid layer may be a film. In some cases, the fluid layer may have a thickness of at least about 10 nanometers (nm), 20nm, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 600nm, 700nm, 800nm, 900nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm or more. Alternatively or additionally, the fluid layer may have a thickness of at most about 1mm, 900 μm, 800 μm, 700 μm, 600 μm, 500 μm, 400 μm, 300 μm, 200 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, 19 μm, 18 μm, 17 μm, 16 μm, 15 μm, 14 μm, 13 μm, 12 μm, 11 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm, 900mm, 800mm, 700mm, 600mm, 500nm, 450nm, 400nm, 350nm, 300nm, 250nm, 200nm, 150nm, 100nm, 90nm, 80nm, 70nm, 60nm, 50nm, 40nm, 30nm, 20nm, 10nm or less. Alternatively or additionally, the thickness of the fluid layer may be between any range of any two of the foregoing values.

Fig. 2A shows a partial cross-sectional view of a barrier system 200 holding a fluid barrier 213. Fig. 2B shows a reduced view of the barrier system 200. Fig. 2C shows a perspective view of the chamber 215 of the barrier system 200. Barrier system 200 and/or its respective components may correspond to barrier system 100 and/or its respective components.

Barrier system 200 includes a sample environment 205 defined by a plate 203, a chamber 215, and a fluid barrier 213. The chamber 215 and the plate 203 may be separated by a physical gap. The sample environment 205 may be isolated (and/or insulated) from the external environment 207.

The fluid barrier 213 may act as a transition region between the sample environment 205 and the external environment 207. The fluid barrier 213 may include a fluid (e.g., air) from the sample environment 205, the external environment 207, or both. The fluid barrier 213 may be a low pressure region. The fluid barrier 213 may have a lower pressure than the sample environment, the external environment, or both. The fluid barrier 213 may be maintained by a fluid flow unit such as a pressure changing device 211. The fluid barrier 213 may comprise a fluid that is in coherent or bulk motion.

The pressure changing device 211 may be integrated with the chamber 215. For example, as shown in fig. 2A-2C, the pressure changing device may be integrated as a fluid channel 220 in the wall of the chamber 215. For example, suction may be applied through the fluid channel 220 to draw fluid from the external environment 207 or the sample environment 205 or both to create a partial vacuum curtain (e.g., in coherent motion, in bulk motion, etc.), thereby forming the fluid barrier 213. Otherwise, the fluid may be subjected to negative pressure. The fluid discharge may be discharged at the other end of the fluid channel. Alternatively or additionally, the device may not be integrated with the chamber 215. The fluid flow unit and/or pressure changing device 211 may be operated by one or more compressors (e.g., to generate negative pressure), pumps (e.g., to generate positive pressure), suction equipment, and/or other devices to provide a lower pressure in the transition region. The chamber 215 may include one or more fluid channels 220 for implementing the fluid barrier of the present disclosure.

While two pressure changing devices 211 are shown in fig. 2A-2C, it should be understood that any number of such devices may be present. For example, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more such devices may be present. Alternatively or additionally, up to about 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2 such devices may be present. In some cases, one or more of the pressure changing devices 211 may be implemented as an annular fluid channel around the sample environment area, or other fluid channel along the perimeter or boundary of the sample environment area. In some cases, one or more additional fluid flow channels (e.g., 233) may be provided near the bottom of the chamber to draw excess fluid (e.g., liquid, gas) from the sample environment area.

Advantageously, the fluid barrier 213 may provide a low friction or zero friction seal between the sample environment 205 and the external environment 207. In some cases, the fluid flow rate through the fluid barrier 213 may be at least about 5 liters per minute (L/min), 5.5L/min, 6L/min, 6.5L/min, 7L/min, 7.5L/min, 8L/min, 8.5L/min, 9L/min, 9.5L/min, 10L/min, 10.5L/min, 11L, 11.5L/min, 12L/min, 12.5L/min, 13L/min, 13.5L/min, 14L/min, 14.5L/min, 15L/min, or more. Alternatively or additionally, the fluid flow rate may be up to about 15L/min, 14.5L/min, 14L/min, 13.5L/min, 13L/min, 12.5L/min, 12L/min, 11.5L/min, 11L/min, 10.5L/min, 10L/min, 9.5L/min, 9L/min, 8.5L/min, 8L/min, 7.5L/min, 7L/min, 6.5L/min, 6L/min, 5.5L/min, 5L/min, or less. It should be appreciated that the fluid flow rate may vary with different parameters (e.g., minimum distance between plate and chamber, pressure, temperature, etc.). In some examples, the fluid flow rate may be about 10L/min or about 13 milliliters/minute (mL/min)/millimeter (mm) along a peripheral velocity of about 0.42 meters/second (m/s) for a gap of about 500 microns between the plate 203 and the chamber 215.

The system of the present disclosure may be scaled, for example, to have multiple sample environment areas defined by the same plate. Fig. 3 illustrates a barrier system 300 having multiple sample environments. Barrier system 300 and/or its respective components may correspond to any other barrier system (e.g., 100 and/or 200) and/or its respective component described herein.

The single plate 303 may define at least two separate sample environments 305, 309, which are further defined by two separate chambers. Each sample environment may be controlled and maintained independently of the other sample environments. Each sample environment is movable relative to the plate 303 independently of the other sample environments. A fluid barrier may be maintained between each sample environment and the external environment.

Although two sample environments are shown in fig. 3, it should be understood that the system of the present disclosure may be implemented for any number of sample environments using a single plate. For example, there may be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more such sample environments in a single plate system. Alternatively or additionally, there may be up to about 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2 such sample environments. Any subset or all of the plurality of sample environments may be capable of moving independently of the other sample environments.

In some cases, a single detector in plate 303 may be used to detect one or more sample environments. Alternatively or additionally, a single plate 303 may allow at least two detectors to protrude through the single plate 303 for parallel detection. For example, such a detector may protrude through the plate via one or more holes 321a, 321b, the holes 321a, 321b having a fluid tight fit with the detector. The detector may be fixed relative to the plate. In some cases, multiple detectors may detect two different locations in the same sample environment in parallel. In some cases, multiple detectors may detect at least two different sample environments in parallel.

Although two detector apertures are shown in fig. 3, it should be understood that the system of the present disclosure may be implemented for any number of detectors using a single plate. For example, there may be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more detectors in a single plate system. Alternatively or additionally, there may be up to about 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2 such detectors.

The sample environment (e.g., 105, 205, 305, 309) of the present disclosure may be controlled. For example, the environment may be maintained at a specified temperature or humidity. The environment (or any element thereof) may be maintained at a temperature of at least about 20 degrees celsius (c), 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃, 85 ℃, 90 ℃, 95 ℃, 100 ℃, or more. Alternatively, the environment may be maintained below 20 ℃. Alternatively or additionally, the environment (or any element thereof) may be maintained at a temperature of up to about 100 ℃, 95 ℃, 90 ℃, 85 ℃, 80 ℃, 75 ℃, 70 ℃, 65 ℃, 60 ℃, 55 ℃, 50 ℃, 45 ℃, 40 ℃, 35 ℃, 30 ℃, 25 ℃, 20 ℃ or less. The environment may be maintained at a temperature within a range defined by any two of the foregoing values.

Different elements of the sample environment, such as the chambers therein, protruding portions of the detector, immersion fluid, plates, substrates, solutions, and/or samples may be maintained at different temperatures or within different temperature ranges, such as the temperatures or temperature ranges described herein. The temperature of the system components may be set at a temperature above the dew point to prevent condensation. The components of the system may be set at a temperature below the dew point to collect condensed water.

In some cases, the sample environment may be maintained at a higher humidity than the external environment. In some cases, the sample environment may be maintained at a relative humidity of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. Alternatively or additionally, the relative humidity may be maintained at up to about 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or less. Alternatively or additionally, the relative humidity may be maintained within a range defined by any two of the foregoing values.

An environmental unit (e.g., humidifier, heater, heat exchanger, compressor, etc.) may be configured to regulate one or more operating conditions in each sample environment. In some cases, each environment may be regulated by a separate environmental unit. In some cases, a single environmental unit may regulate multiple environments. In some cases, multiple environmental units may individually or collectively regulate different environments. The environmental unit may use either active or passive methods to adjust the operating conditions. For example, a heating element or a cooling element may be used to control the temperature. A humidifier or dehumidifier may be used to control humidity.

In some cases, a first portion of the sample environment may be further controlled by other portions of the sample environment. Different local components may have different local temperatures, pressures, and/or humidities. For example, the sample environment may comprise a first internal or local environment and a second internal or local environment, e.g. separated by a seal. In some cases, the seal may include an immersion objective, as described elsewhere herein. For example, the immersion objective may be part of a seal that separates the sample environment into a first internal environment having 100% (or substantially 100%) relative humidity and a second environment having a different temperature, pressure, or humidity. The second environment may or may not be an ambient environment. The immersion objective may be in contact with the detector.

Fig. 4 illustrates an exemplary barrier system 400 including different local environments 441, 442, 443, showing a cross-sectional side view. The example barrier system 400 and one or more components therein may correspond to the example barrier system 100 and one or more components therein. A fluid barrier 413 may be implemented between the sample environment 405 (e.g., the first region) and the external environment (e.g., the second region). The sample environment 405 may be a controlled environment including one or more samples. The external environment may be a closed environment or an open environment. The sample environment 405 area may be defined by a chamber 415, a plate 403, and a fluid barrier 413. The fluid barrier 413 may be maintained between the physical gap between the chamber 415 and the plate 403. Chamber 415 and plate 403 may be independent such that chamber 145 and the sample environment 405 area defined thereby are movable relative to plate 403. The plate 403 and the chamber 415 may not be in direct mechanical contact such that there is a minimum distance between the plate and the chamber. The fluid barrier 413 may include fluid from the sample environment, the external environment, or both, and act as a transition region between the sample environment 405 and the external environment.

Sample environment 405 may include a substrate 417. One or more samples may be immobilized on base 417 or adjacent to base 417. Alternatively or additionally, one or more samples may be otherwise disposed on base 417. In some cases, at least a portion of chamber 415 may be or include base 417. In other cases, chamber 415 may be coupled to base 417. In some cases, base 417 may be fixed relative to chamber 415. Alternatively, base 417 may be movable relative to chamber 415, such as in a linear and/or non-linear (e.g., rotational) direction. For example, base 417 may be fixed to chamber 415 in XY coordinates (and/or Z coordinates) but rotatable relative to chamber 415.

The detector 401 may protrude from the external environment into the sample environment 105 through the plate 403, for example through a hole in the plate 403. At least a portion of the detector 401 may be fixed relative to the plate 403. In some cases, detector 401 may be capable of translating independently of plate 403 along an axis substantially perpendicular to the plane of plate 403 (e.g., through the aperture). Within sample environment 405, detector 401 may be configured to detect one or more samples disposed on a substrate using an immersion optical system, such as the system described with respect to fig. 1C. A portion of the detector within the sample environment 401, such as an optical imaging objective, may be in optical communication with the substrate through a liquid fluid 431 medium. In some cases, the liquid fluid medium may be disposed on a localized area of the base 417. Alternatively, the detector 401 may be in optical communication with the substrate in the absence of a liquid fluid medium.

The sample environment 405 may include any number of different local environments 441, 442, 443 located at different portions of the sample environment. Different local environments may be adjusted. The fluid barrier 413 may maintain different local environments under different local environmental conditions. For example, the local environment may be maintained at a local temperature or a local temperature range. For example, the local environment may be maintained at a local humidity or a local humidity range. For example, the local environment may be maintained at a local pressure or local pressure range. The local temperature may be at least about 20 degrees celsius (°c), 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃, 85 ℃, 90 ℃, 95 ℃, 100 ℃, or more. Alternatively, the local temperature may be maintained below 20 ℃. Alternatively or additionally, the local temperature may be up to about 100 ℃, 95 ℃, 90 ℃, 85 ℃, 80 ℃, 75 ℃, 70 ℃, 65 ℃, 60 ℃, 55 ℃, 50 ℃, 45 ℃, 40 ℃, 35 ℃, 30 ℃, 25 ℃, 20 ℃ or less. The local environment may be maintained at a local temperature within a range defined by any two of the foregoing values. The local relative humidity may be at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. Alternatively or additionally, the local relative humidity may be up to about 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or less. Alternatively or additionally, the relative humidity may be maintained within a range defined by any two of the foregoing values. Any of the environmental units described herein may be used to maintain a local environment.

In the example shown in fig. 4, a first local environment 441 located at or near the top of the sample environment 405 (or chamber 415) is maintained at a highest local temperature within the sample environment 405, for example, to prevent undesirable materials from condensing and dripping onto the substrate 417. The second local environment 442 includes a humidity source 419, such as a pool of liquid (e.g., water), at or near the bottom of the sample environment 405 (or chamber 415). The second local environment 442 may be maintained at a second highest local temperature within the sample environment 405, for example, to generate steam from the humidity source 419. The third local environment 443 is at or near the surface of the substrate 417 and is maintained at a minimum local temperature within the sample environment 405, for example, to prevent the surface from drying out. Although three local environments are shown, it should be appreciated that the sample environment may have any number of different local environments maintained under different local environmental conditions, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more local environments.

The objective lens of detector 401 may be heated to prevent condensation and disruption of the optical path between the objective lens and substrate 417. Alternatively or additionally, another component or part of the detector in fluid contact with the substrate may be heated. As used herein, the term "heating" may generally refer to subjecting an object to an increase in temperature compared to a reference temperature prior to a heating operation. Heating may be performed by the environmental unit described herein. Heating may be performed by heating or maintaining the temperature (or range thereof) of a volume of immersion fluid in contact with the detector and the substrate. The heating element may be coupled or integrated with the objective lens (and/or other components of the detector).

There is provided a system for processing or analyzing an analyte, comprising: a chamber and a lid. The chamber may include an interior region including a substrate configured to immobilize an analyte immobilized thereon. The cover may be configured to be disposed adjacent to the chamber. The system may include an environmental unit configured to maintain a first local environment, a second local environment, and a third local environment within the interior region. The environmental unit may be configured to maintain the first local environment at a first temperature or temperature range, maintain the second local environment at a second temperature or temperature range, and maintain the third local environment at a third temperature or temperature range. The first local environment may be disposed above the second local environment and the third local environment. The first local environment may be at or near the cover. The second local environment may be disposed at or near the substrate surface. The third local environment may be disposed under the first local environment and the second local environment. The first temperature or temperature range may be higher than the second temperature or temperature range and the third temperature or temperature range. The second temperature or temperature range may be lower than the third temperature or temperature range.

Fig. 5 illustrates a processing system 500 including an exemplary barrier system. The processing system 500 may include one or more modular components.

The processing system 500 may include one or more barrier systems, such as a first barrier system 505a and a second barrier system 505b. The barrier systems (e.g., 505a, 505 b) of the processing system 500 and one or more components thereof may correspond to any of the barrier systems and one or more components thereof described herein. Although two barrier systems are shown in fig. 5, it should be understood that the processing system may have any number of barrier systems.

In some cases, any barrier system of the present disclosure may be used to handle operations in lieu of or in addition to detection.

For example, any barrier system of the present disclosure may have one or more alternative detectors (e.g., 501) or an operating unit other than a detector. The manipulation unit may comprise one or more devices or components thereof and is configured to facilitate manipulation with respect to a sample or sample environment (or one or more local environments thereof). For example, the operating unit may comprise one or more detectors configured to facilitate detection of a signal or signal change from the sample. In another example, the manipulation unit can include a fluid dispenser (e.g., 509a, 509 b) configured to facilitate dispensing of a reagent or fluid to the sample. In another example, the manipulation unit may include an environmental unit configured to facilitate environmental conditioning of the sample environment. In another example, the operating unit may include a light source, a heat source, or a humidity source. In another example, the operating unit may include any one or more sensors. The barrier system may have a plurality of operating units of the same or different types.

The manipulation unit (e.g., 509 a) may protrude from the external environment into the sample environment of the barrier system through a plate (e.g., 503), such as through a hole in the plate. The fit between the operating unit and the aperture may be fluid tight such that there is no fluid communication through the aperture when the operating unit is assembled through the aperture. Alternatively or additionally, the aperture may be hermetically sealed. Alternatively, the plate may be integrated with the operation unit, or the operation unit may be integrated with the plate. Alternatively, the operating unit may be fully contained in the sample environment, for example by fixing the non-sample-facing end to the plate. In some cases, at least a portion of the operating unit may be fixed relative to the plate. In some cases, the operating unit may be translatable independently of the plate along an axis substantially perpendicular to the plane of the plate (e.g., through the aperture). In some cases, at least a portion of the operating unit (e.g., a portion of the operating unit within the sample environment region) may be capable of moving (e.g., linearly or non-linearly, e.g., rotating) independent of the plate.

In some cases, the processing system 500 may include a plurality of modular plates (e.g., 503a, 503b, 503 c) that may be coupled or otherwise fastened to each other to form the uninterrupted plate 503. The fit between adjoining modular plates may be fluid tight such that there is no fluid communication between the modular plates. Alternatively or additionally, the mating may include a hermetic seal. Adjoining modular plates (e.g., a first modular plate and a second modular plate) may be coupled by one or more fastening mechanisms. Examples of fastening mechanisms may include, but are not limited to, complementary threads, form-fit pairs, shackles, latches, threads, screws, locking rings, clips, clamps, prongs, rings, tacks, rubber bands, rivets, grommets, pins, wire bands, buttons, velcro, adhesives (e.g., glue), tape, vacuum, seals, magnets, magnetic seals, combinations thereof, or any other type of fastening mechanism.

In some cases, the first and second modular plates may be fastened to each other by complementary fastening units. For example, the first modular plate and the second modular plate may complete a form fit pair. The first modular plate may comprise a positive-fit male component and the second modular plate may comprise a positive-fit female component, and/or vice versa. In some cases, the outer diameter of the protruding fastening unit of the first modular plate may be substantially equal to the inner diameter of the recessed fastening unit of the second modular plate, or vice versa, to form an interference fit. Alternatively or additionally, the two modular plates may include other types of complementary units or structures (e.g., shackle, latch, snap, button, nut and bolt, magnet, etc.) that may be fastened together. Alternatively or additionally, the two modular plates may be fastened using other fastening mechanisms such as, but not limited to, staples, clips, jaws, prongs, rings, tacks, rubber bands, rivets, grommets, pins, straps, buttons, velcro, adhesives (e.g., glue), magnets or magnetic fields, tape, combinations thereof, or any other type of fastening mechanism.

In some cases, the first modular plate and the second modular plate may be secured to each other by an intermediate structure. The intermediate structure may be a link or a connection between the first modular plate and the second modular plate. In some cases, the intermediate structure may be secured to one or both of the first and second modular plates by one or more of any of the securing mechanisms described herein. The intermediate structure may be solid. The intermediate structure may be a liquid or a gas. The intermediate structure may be a gel. In some cases, the intermediate structure may be applied as one phase (e.g., liquid) and transition to another phase (e.g., solid) after the passage of time, for example, to achieve fastening. For example, the intermediate structure may include a fluid adhesive that cures to achieve a secure. In some cases, upon application of a stimulus (e.g., a thermal change, a pH change, a pressure change, a magnetic field, an electric field, etc.) to effect tightening or loosening (or both), the intermediate structure may be able to transition from a first phase to a second phase, such as from a liquid to a solid or from a solid to a liquid. In some cases, the first modular plate and/or the second modular plate may comprise an intermediate structure. For example, the intermediate structure may be integrated with the first modular plate and/or the second modular plate. In some cases, a first modular plate and/or a second modular plate may be partially or entirely capable of transitioning from a first phase to a second phase, such as from a liquid to a solid or from a solid to a liquid, upon application of a stimulus (e.g., a thermal change, a pH change, a pressure change, a magnetic field, an electric field, etc.) to effect fastening or unfastening (or both) with another plate. In some cases, when two modular plates are fastened together, one or both of the two modular plates may be cut or pierced by the other.

The fastening between the first and second modular plates may be temporary, e.g., allowing subsequent loosening of the two modular plates to the two modular plates without damage (e.g., permanent deformation, defect, etc.) or with minimal damage. In some cases, the first modular plate may be able to be repeatedly and easily released from the second modular plate and/or the rest of the plate 503.

In some cases, a modular plate may be disengaged from another modular plate or the remainder of the plate without interfering with one or more sample environments of a respective one or more barrier systems comprising at least a portion of the remainder of the plate, such as during operation by one or more operating units (e.g., reagent dispensing, washing, detection, etc.) of the one or more barrier systems. Advantageously, the disengagement of the modular plates may allow access to a chamber, such as a chamber in the load or unload processing system 500. The disengagement of the modular plates may also allow access to the interior of the chambers of the barrier system, such as loading or unloading substrates from the chambers. The disengagement of the modular plate may also allow access to one or more operating units coupled to or otherwise associated with the disengaged modular plate, e.g., for maintenance, repair, and/or replacement of one or more operating units. Such detachment may occur when another barrier system is subjected to normal operation (e.g., chemical treatment operation, detection operation, etc.). In some cases, the disengagement of the modular plates may be along the Z-axis or substantially the Z-axis, or along any other axis (e.g., X-Y plane, etc.). In some cases, any modular plate may be disengaged from another modular plate. In some cases, any modular plate may be movable relative to another modular plate. In some cases, any modular plate may move relative to the reference coordinate during disengagement. In some cases, any modular plate may be substantially stationary relative to the reference coordinate during disengagement. In some cases, a first modular plate (e.g., 520a, 520c, etc.) may be movable and a second modular plate (e.g., 520 b) may be stationary relative to the reference coordinates.

The processing system 500 may include different stations (e.g., 520a, 502b, 520 c). The operator station may be positioned relative to a portion of the plate 503. In some cases, a single modular plate may include one or more operating units for operating the station. In some cases, the plurality of modular plates may include one or more operating units for operating the station. In some cases, a single modular plate may include one or more operating units for multiple operating stations. In some cases, the plurality of modular boards may include one or more operating units for the plurality of operating stations. The stations may include chemical stations (e.g., 520a, 520 c), for example, for reagent dispensing and/or washing. The operator stations may include detection stations (e.g., 520 b), for example, for detecting signals or signal changes. Any barrier system (e.g., 505a, 505 b) of the processing system may be capable of traveling between different stations. Alternatively or additionally, the plate 503 may be capable of traveling relative to any barrier system to position the barrier system relative to the operator station (e.g., positioned relative to a portion of the plate). In some cases, the barrier system may provide rails or tracks 507 or other motion paths to allow travel between different operator stations. In some cases, different obstacle systems may share the same rail or track or other path of movement for travel between different operating systems (e.g., as shown in fig. 5). In this case, the different barrier systems may be configured to move independently of each other, or in unison, on the same rail or track or other path of motion. In some cases, different barrier systems may move on dedicated, individual rails or tracks or other paths of motion. In some cases, the fluid barrier of the barrier system may be maintained during relative movement between the plate 503 and the barrier system, for example during switching of the operating station. In some cases, one or more of the operating units may be movable relative to the plate 503 (e.g., along an axis perpendicular to the plate) or removable from the plate 503 to allow the barrier system to be positioned relative to the operating station.

The external environment (e.g., 107, 207) of the present disclosure may be any environment other than the sample environment. For example, the external environment may be an indoor environment. The external environment may be an ambient environment. The external environment itself may be controlled, for example, via one or more environmental units described elsewhere herein. The external environment may be open or closed. In some cases, the external environment may be at room temperature, pressure, and/or humidity. In some cases, the external environment may be at ambient temperature, pressure, and/or humidity.

The chambers (e.g., 115, 215, 415) of the present disclosure may include a base and sidewalls to define an opening that nearly contacts the plate (or lid). The sidewall may be a closed continuous surface, or a plurality of adjacent (and/or abutting) surfaces. For example, the base may comprise or be a substrate. In some cases, the base may be coupled to the substrate. The base may be translationally fixed to the base. The base may be rotatable relative to the base. The translational movement may comprise a movement of the object from the first coordinate to the second coordinate. The translational movement may comprise a movement of the reference point of the object from the first coordinate to the second coordinate. In some cases, at least a portion of the sidewall of the chamber may have a thickness dimension large enough to allow integration of one or more fluid channels to allow operation of the pressure changing device. In some cases, the sidewall of the chamber may have a thickness dimension large enough to maintain a low pressure fluid barrier. The chamber may comprise, in whole or in part, glass, silicon, a metal such as aluminum, copper, titanium, chromium, or steel, a ceramic such as titanium oxide or silicon nitride, a plastic such as Polyethylene (PE), low Density Polyethylene (LDPE), high Density Polyethylene (HDPE), polypropylene (PP), polystyrene (PS), high Impact Polystyrene (HIPS), polyvinyl chloride (PVC), polyvinylidene chloride (PVDC), acrylonitrile Butadiene Styrene (ABS), polyacetylene, polyamide, polycarbonate, polyester, polyurethane, polyepoxide, polymethyl methacrylate (PMMA), polytetrafluoroethylene (PTFE), phenolic (PF), melamine Formaldehyde (MF), urea Formaldehyde (UF), polyetheretherketone (PEEK), polyetherimide (PEI), polyimide, polylactic acid (PLA), one or more of furan, silicone, polysulfone, any mixture of any of the foregoing, or any other suitable material.

The substrate (e.g., 417) of the present disclosure may be an open substrate. The substrate may be a solid substrate. The substrate may comprise, in whole or in part, glass, silicon, a metal such as aluminum, copper, titanium, chromium, or steel, a ceramic such as titanium oxide or silicon nitride, a plastic such as Polyethylene (PE), low Density Polyethylene (LDPE), high Density Polyethylene (HDPE), polypropylene (PP), polystyrene (PS), high Impact Polystyrene (HIPS), polyvinyl chloride (PVC), polyvinylidene chloride (PVDC), acrylonitrile Butadiene Styrene (ABS), polyacetylene, polyamide, polycarbonate, polyester, polyurethane, polyepoxide, polymethyl methacrylate (PMMA), polytetrafluoroethylene (PTFE), phenolic (PF), melamine Formaldehyde (MF), urea Formaldehyde (UF), polyetheretherketone (PEEK), polyetherimide (PEI), polyimide, polylactic acid (PLA), one or more of furan, silicone, polysulfone, any mixture of any of the foregoing, or any other suitable material. The substrate may be coated in whole or in part with one or more layers of a metal (such as aluminum, copper, silver, or gold), an oxide (such as silicon dioxide) (Si _x O _y Where x, y may take any possible value), photoresist (such as SU 8), surface coating (such as aminosilane or hydrogel), polyacrylic acid, polyacrylamide dextran, polyethylene glycol (PEG), or any combination of any of the foregoing, or any other suitable coating. One or more layers may have at least 1 nanometer (nm), at least 2nm, at least 5nm, at least 10nm, at least 20nm, at least 50nm, at least 100nm, at least 200nm, at least 500nm, at least 1 micrometer (μm), at least 2 μm, at least 5 μm, at least 10 μm, at least 20 μm, at least 50 μm, at least 100 μm, at least 200 μm, at least 500 μm, or at least 1 millimeter (mm) Thickness. One or more layers may have a thickness within a range defined by any two of the foregoing values.

The substrate and/or chamber may have any shape, form or size. In some cases, for example, the substrate may have the general form of a cylinder, cylindrical shell or disk, rectangular prism, or any other geometric shape. The substrate can have a thickness (e.g., minimum dimension) of at least about 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 1mm, 2mm, 3mm, 4mm, 5mm, 1cm, 2cm, 3cm, 4cm, 5cm, or more. The substrate may have a thickness within a range defined by any two of the foregoing values. The substrate can have a first lateral dimension (e.g., a width of the substrate having a general shape of a rectangular prism or a radius of the substrate having a general shape of a cylinder) of at least about 1mm, 2mm, 3mm, 4mm, 5mm, 1cm, 2cm, 3cm, 4cm, 5cm, 6cm, 7cm, 8cm, 9cm, 10cm, 20cm, 30cm, 40cm, 50cm, 60cm, 70cm, 80cm, 90cm, 1 meter (m) or more. The substrate may have a first transverse dimension within a range defined by any two of the foregoing values. The substrate may have a second transverse dimension (e.g., the length of a substrate having the general shape of a rectangular prism) or at least about 1mm, 2mm, 3mm, 4mm, 5mm, 1cm, 2cm, 3cm, 4cm, 5cm, 6cm, 7cm, 8cm, 9cm, 10cm, 20cm, 30cm, 40cm, 50cm, 60cm, 70cm, 80cm, 90cm, 1 meter (m), or more. The substrate may have a second transverse dimension within a range defined by any two of the foregoing values. The surface of the substrate may be planar or substantially planar. Alternatively or additionally, the surface of the substrate may be textured or patterned. For example, the substrate may include grooves, slots, ramps, and/or posts. In some cases, the substrate may include holes. In some cases, the substrate may define one or more cavities (e.g., microscale cavities or nanoscale cavities). The substrate may have a regular texture and/or pattern over the entire surface of the substrate. For example, the substrate may have a regular geometry (e.g., wedge, cuboid, cylinder, sphere, hemisphere, etc.) above or below a reference level of the surface. Alternatively, the substrate may have an irregular texture and/or pattern on the entire surface of the substrate. For example, the substrate may have any arbitrary structure above or below a reference level of the substrate. In some cases, the texture of the substrate may include structures having a maximum dimension of at most about 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001% of the total thickness of the substrate or substrate layer. In some cases, the texture and/or pattern of the substrate may define at least a portion of the individually addressable locations on the substrate. The textured and/or patterned substrate may be substantially planar.

The substrate may comprise an array. For example, the array may be located on a side surface of the substrate. The array may be a planar array. The array may have a general shape that is circular, annular, rectangular, or any other shape. The array may comprise linear and/or non-linear rows. The arrays may be evenly spaced or distributed. The arrays may be arbitrarily spaced or distributed. The array may have a regular pitch. The array may have an irregular pitch. The array may be a texture array. The array may be a patterned array. Fig. 6 shows an example (e.g., from a top view) of an array of individually addressable locations 601 on a substrate, panel a of fig. 6 shows a substantially rectangular substrate with a regular linear array, panel B of fig. 6 shows a substantially circular substrate with a regular linear array, and panel C of fig. 6 shows an arbitrarily shaped substrate with an irregular array.

The array may include a plurality of individually addressable locations (e.g., 501). In some cases, the locations may correspond to individually addressable coordinates on the substrate. Alternatively or additionally, the locations may correspond to physical structures (e.g., holes) on the substrate. Analytes to be processed and/or detected by the detector may be immobilized to the array. The array may comprise one or more of the adhesives described herein, such as one or more physical links or linkers or chemical linkers or linkers, coupled or configured to be coupled with the analyte. For example, the array may comprise a linker or adapter that is coupled to the nucleic acid molecule. Alternatively or additionally, the analyte may be coupled to a bead, and the bead may be immobilized to the array.

The individually addressable locations may include locations of analytes or groups of analytes that are available for manipulation. Manipulation may include placement, extraction, reagent dispensing, inoculation, heating, cooling, or agitation. Extraction may include extracting a single analyte or group of analytes. For example, extracting may include extracting at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, or at least 1,000 analytes or groups of analytes. Alternatively or additionally, the extracting may comprise extracting at most 1,000, at most 500, at most 200, at most 100, at most 50, at most 20, at most 10, at most 5, or at most 2 analytes or groups of analytes. Manipulation may be accomplished by, for example, local microfluidic, pipette, optical, laser, acoustic, magnetic, and/or electromagnetic interactions with the analyte or its surroundings.

The array may be coated with an adhesive. For example, the array may be randomly coated with adhesive. Alternatively, the array may be coated with adhesive arranged in a regular pattern (e.g., in a linear array, radial array, hexagonal array, etc.). The array may be coated with the adhesive over at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the total number of individually addressable locations or the surface area of the substrate. The array may be coated with adhesive on a portion of the individually addressable locations or a portion of the surface area of the substrate, the portion being within a range defined by any two of the foregoing values. The adhesive may be integral to the array. An adhesive may be added to the array. For example, the adhesive may be added to the array as one or more coatings on the array.

The binder may immobilize the analyte through non-specific interactions, such as one or more of hydrophilic interactions, hydrophobic interactions, electrostatic interactions, physical interactions (e.g., adhesion to the column or sedimentation within the well), and the like. In some cases, the binding agent may immobilize the biological analyte through a specific interaction. For example, where the biological analyte is a nucleic acid molecule, the adhesive may comprise an oligonucleotide adapter configured to bind the nucleic acid molecule. Alternatively or additionally, the binding agent may comprise one or more of antibodies, oligonucleotides, aptamers, affinity binding proteins, lipids, carbohydrates, etc., for example, for binding to other types of analytes. The binding agent may immobilize the biological analyte by any possible combination of interactions. For example, the binding agent may immobilize the nucleic acid molecule by a combination of physical and chemical interactions, by a combination of protein and nucleic acid interactions, or the like. The array may include at least about 10, 100, 1000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000, or more binders. Alternatively or additionally, the array may comprise up to about 100,000,000, 10,000,000, 1,000,000, 100,000, 10,000, 1000, 100, 10, or less binder. The array may have a plurality of adhesives within a range defined by any two of the foregoing values. In some cases, a single binder may bind a single analyte (e.g., a nucleic acid molecule). In some cases, a single binder can bind multiple analytes (e.g., multiple nucleic acid molecules). In some cases, multiple binders may bind a single analyte. Although some examples herein describe interactions of the adhesive with nucleic acid molecules, the adhesive may immobilize other molecules (e.g., proteins), other particles, cells, viruses, other organisms, etc., as well as non-biological analytes.

In some cases, each location or a subset of such locations may have an analyte (e.g., a nucleic acid molecule, a protein molecule, a carbohydrate molecule, etc.) immobilized thereon. In other cases, a portion of the plurality of individually addressable locations may have an analyte immobilized thereon. The plurality of analytes immobilized on the substrate may be copies of the template analytes. For example, multiple analytes (e.g., nucleic acid molecules) may have sequence homology. In other cases, the plurality of analytes immobilized on the substrate may not be copies. The plurality of analytes may be the same type of analyte (e.g., a nucleic acid molecule) or may be a combination of different types of analytes (e.g., a nucleic acid molecule, a protein molecule, etc.).

In some cases, the array may include multiple types of binders, such as to bind different types of analytes. For example, the array may include a first type of adhesive (e.g., an oligonucleotide) configured to bind a first type of analyte (e.g., a nucleic acid molecule) and a second type of adhesive (e.g., an antibody) configured to bind a second type of analyte (e.g., a protein), and so forth. In another example, the array can include a first type of adhesive that binds a first type of nucleic acid molecule (e.g., a first type of oligonucleotide molecule) and a second type of adhesive that binds a second type of nucleic acid molecule (e.g., a second type of oligonucleotide molecule), and so forth. For example, the substrate may be configured to bind different types of analytes in certain portions or specific locations of the substrate by having different types of adhesives in certain portions or specific locations of the substrate.

The analyte may be immobilized to the array at a given individually addressable location of the plurality of individually addressable locations. The array may have any number of individually addressable locations. For example, the array may have at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, at least 50,000, at least 100,000, at least 200,000, at least 500,000, at least 1,000,000, at least 2,000,000, at least 5,000,000, at least 10,000,000, at least 20,000, at least 50,000,000, at least 100,000,000, at least 200,000, at least 500,000, at least 1,000,000,000, at least 2,000,000, at least 5,000,000,000, at least 10,000,000,000, at least 20,000,000,000, at least 50,000,000,000, or at least 100,000,000,000,000. The array may have a plurality of individually addressable locations that are within a range defined by any two of the foregoing values. Each individually addressable location may be digitally and/or physically accessible (from a plurality of individually addressable locations). For example, each individually addressable location may be located, identified, and/or accessed electronically or digitally for mapping, sensing, association with a device (e.g., detector, processor, dispenser, etc.), or otherwise processed. Alternatively or additionally, each individually addressable location may be physically located, identified, and/or accessed, such as for physical manipulation or extraction of analytes, reagents, particles, or other components located at the individually addressable location.

Each individually addressable location may have the general shape or form of a circle, rectangle, pit, bump, or any other shape or form. Each individually addressable location may have a first lateral dimension (such as a radius for an individually addressable location having a circular general shape or a width for an individually addressable location having a rectangular general shape). The first lateral dimension may be at least 1 nanometer (nm), at least 2nm, at least 5nm, at least 10nm, at least 20nm, at least 50nm, at least 100nm, at least 200nm, at least 500nm, at least 1,000nm, at least 2,000nm, at least 5,000nm, or at least 10,000nm. The first transverse dimension may be within a range defined by any two of the foregoing values. Each individually addressable location may have a second lateral dimension (such as for the length of an individually addressable location having the general shape of a rectangle). The second lateral dimension may be at least 1 nanometer (nm), at least 2nm, at least 5nm, at least 10nm, at least 20nm, at least 50nm, at least 100nm, at least 200nm, at least 500nm, at least 1,000nm, at least 2,000nm, at least 5,000nm, or at least 10,000nm. The second transverse dimension may be within a range defined by any two of the foregoing values. In some cases, each individually addressable location may have an adhesive as described herein or be coupled to an adhesive as described herein to immobilize an analyte thereto. In some cases, only a portion of the individually addressable locations may have an adhesive or be coupled with an adhesive. In some cases, the individually addressable locations may have or be coupled with multiple adhesives to immobilize the analyte thereto.

Analytes bound to the individually addressable locations may include, but are not limited to, molecules, cells, organisms, nucleic acid molecules, nucleic acid colonies, beads, clusters, populations, or DNA nanospheres. The bound analytes may be immobilized to the array in a regular, patterned, periodic, random or pseudo-random configuration or any other spatial arrangement.

While the examples of the present disclosure describe the processing and/or detection of samples and analytes fixed to individually addressable locations on a substrate, the systems, devices, and methods described herein also allow for the detection of the substrate itself (without any samples and/or analytes disposed thereon).

The substrate may be configured to move relative to the plate. Such movement may be facilitated by one or more actuators or other devices (e.g., gears, stages, actuators, discs, pulleys, motors, etc.). Such actuators and devices may be mechanically coupled to the substrate directly or indirectly through an intermediate assembly. Such actuators and devices may be automated. Alternatively or additionally, the actuator and device may receive manual inputs. The substrate may be configured to move at any speed that allows detection. In some cases, or rotational movement, the axis of rotation may be an axis passing through the center of the substrate. The shaft may be an eccentric shaft. For example, the substrate may be secured to a chuck (e.g., a vacuum chuck). For example, the substrate may be attached to a chuck (such as a vacuum chuck) of a spin coating apparatus. The substrate may be configured to rotate at a rotational speed of at least 1 revolution per minute (rpm), at least 2rpm, at least 5rpm, at least 10rpm, at least 20rpm, at least 50rpm, at least 100rpm, at least 200rpm, at least 500rpm, at least 1,000rpm, at least 2,000rpm, at least 5,000rpm, or at least 10,000 rpm. The substrate may be configured to rotate at a rotational speed within a range defined by any two of the foregoing values. The substrate may be configured to rotate at different rotational speeds during different operations described herein. The substrate may be configured to rotate at a rotational speed that varies according to a time-dependent function, such as a ramp function, a sinusoidal function, a pulsed function, or other function or combination of functions. The time-varying function may be periodic or aperiodic.

The fluid barrier provided herein may provide zero friction or low friction relative motion between the substrate and the detector. There may be no mechanical contact between the plate (coupled to the detector) and the chamber (coupled to the substrate).

The detectors (e.g., 101, 1110) of the present disclosure may include a device capable of detecting a signal. For example, the signal may be a signal indicative of the presence or absence of one or more components (e.g., incorporated nucleotides, fluorescent labels, electronic signals, etc.) and/or a signal indicative of a change in state of one or more components. The detector may detect a plurality of signals. The signal or signals may be detected in real time before, during (or substantially during) or after a reaction, such as a sequencing reaction. In some cases, the detector may include optical and/or electronic components that may detect the signal. The detector may implement one or more detection methods. Non-limiting examples of detection methods include optical detection, spectroscopic detection, electrostatic detection, electrochemical detection, acoustic detection, magnetic detection, and the like. Optical detection methods include, but are not limited to, light absorption, ultraviolet visible (UV-vis) light absorption, infrared light absorption, light scattering, rayleigh scattering, raman scattering, surface enhanced raman scattering, mie scattering, fluorescence, luminescence, and phosphorescence. Spectroscopic detection methods include, but are not limited to, mass spectrometry, nuclear Magnetic Resonance (NMR) spectroscopy, and infrared spectroscopy. Electrostatic detection methods include, but are not limited to, gel-based techniques such as gel electrophoresis. Electrochemical detection methods include, but are not limited to, electrochemical detection of the amplified product after high performance liquid chromatography separation of the amplified product.

A detectable signal, such as an optical signal (e.g., a fluorescent signal), may be generated when the analyte reacts with another component (e.g., a probe). For example, the signal may originate from a probe and/or an analyte. The detectable signal may be indicative of a reaction or interaction between the probe and the analyte. The detectable signal may be a non-optical signal. For example, the detectable signal may be an electronic signal. The detectable signal may be detected by one or more sensors. For example, the optical signal may be detected via one or more optical detectors in an optical detection scheme as described elsewhere herein. The signal may be detected during movement of the substrate. The signal may be detected after the motion is terminated. In some cases, after detection, the signal may be eliminated, such as by cleaving the label from the probe and/or analyte, and/or modifying the probe and/or analyte. Such cleavage and/or modification may be affected by one or more stimuli, such as exposure to chemicals, enzymes, light (e.g., ultraviolet light), or temperature changes (e.g., heat). In some cases, the signal may become undetectable by disabling or changing the mode (e.g., detection wavelength) of one or more sensors, or by terminating or reversing the excitation of the signal. In some cases, detection of the signal may include capturing an image or generating a digital output (e.g., between different images).

The detector may be capable of continuous area scanning during continuous linear motion and/or continuous non-linear (e.g., rotational) motion between the sample and the substrate. For example, the detector may scan the substrate or array along a linear or substantially linear path. Alternatively or additionally, the detector may scan along a non-linear path, including scanning in a loop, spiral, or arc over a rotating substrate. The detector may be a continuous area scanning detector. The continuous area scanning detector may comprise an imaging array sensor capable of continuous integration over the scanning area. The scanning may be electronically synchronized with the image of the relatively moving object. The continuous area scan detector may include a Time Delay and Integration (TDI) Charge Coupled Device (CCD), hybrid TDI, and/or Complementary Metal Oxide Semiconductor (CMOS) pseudo TDI.

For a rotational scan path, the scan direction may be substantially θ in the (R, θ) coordinate system, where the object is in rotational motion in the θ direction. The apparent velocity can be at any field of view on the object (substrate) imaged by the scanning systemThe relation of (2) varies with the radial position (R) of the field point on the object. The continuous area scan detector may scan all image positions at the same rate and thus may not be able to operate on all imaging points in a curved (or arcuate or nonlinear) scan at the correct scan rate. Thus, for imaging field points moving at a different speed than the scan speed, the scan may be affected by the speed blur. The continuous rotation region scan may include an optical detection system or method that performs algorithmic correction, optical correction, and/or electronic correction to substantially compensate for the tangential velocity blur, thereby reducing the scanning aberrations. For example, the compensation is by The differential speed blur at each image location corresponding to a different radius on the rotating substrate is algorithmically implemented using an image processing algorithm that deconvolves the differential speed blur to compensate for the differential speed blur. In another example, compensation is accomplished by using a deformation magnification gradient. This can be used to magnify the substrate by different amounts (deformation magnification) along one axis at two or more substrate positions transverse to the scanning direction. The deformation magnification gradient may modify the imaging speeds of two or more locations to be substantially equal, thereby compensating for tangential speed differences at two locations on the substrate. The compensation may be adjustable to account for different velocity gradients across the field of view at different radii on the substrate. In some cases, the video field imaging may be divided into two or more regions, each of which may be electronically controlled to scan at a different rate. These rates can be adjusted to the average projected object velocity within each region. These regions may be optically defined using one or more beam splitters or one or more mirrors. The two or more regions may be directed to two or more detectors. These regions may be defined as sections of a single detector.

The systems, devices, and methods described herein may have particular biological applications. In some examples, the fluid barrier system may be used in nucleic acid sequencing applications. The sample environment may be provided within a chamber having a substrate comprising an array. Multiple nucleic acid molecules can be immobilized to individually addressable locations in an array. The solution of labeled nucleotides can be dispensed onto a substrate under conditions sufficient to allow at least a subset of the labeled nucleotides to be incorporated into a plurality of nucleic acid molecules of at least a subset, if appropriate (e.g., the labeled nucleotides are complementary to open positions in the nucleic acid molecules), and unincorporated nucleotides are washed with a wash solution. The sample environment, including temperature, pressure, and/or humidity, may be maintained based on the particular sample (e.g., nucleic acid molecule) used in the sample environment and/or the treatment (e.g., incorporation reaction) performed. Then, while achieving a fluid barrier and thereby maintaining sample environmental conditions, a detector (configured as described elsewhere herein) protruding through the plate into the sample environment can detect one or more detectable signals from the incorporated labeled nucleotides at individually addressable locations in the array during relative movement of the detector and the substrate. For example, the substrate may be moved relative to the detector, e.g. to allow the detector to detect all individually addressable locations in the substrate (or desired sub-areas). In some cases, the substrate may be rotated and then linearly moved in a repeating cycle such that after each rotation the detector is able to scan an annular ring and after each linear movement the detector is positioned to scan another annular ring of a different radius from the center of the substrate. Alternatively or additionally, the substrate may be subjected to only rotational movement. Alternatively or additionally, the substrate may perform only linear movements.

The fluid barrier maintained during detection may provide a barrier between the controlled sample environment and the external environment and allow low or zero friction relative movement between the detector and the sample while maintaining a controlled sample environment. Advantageously, such barriers may allow for continuous scanning in a 100% or substantially 100% relative humidity environment. The barrier may prevent moisture from escaping the sample environment, which may condense and affect (e.g., corrode, contaminate, etc.) sensitive equipment, such as optics, when escaping. Furthermore, the barrier may prevent contaminants from the external environment from entering the sample environment, which may affect the fluid and/or detection (e.g., imaging).

It should be understood that the systems, devices, and methods described herein may also have non-biological applications, for example, for analyzing non-biological samples.

Computer system

The present disclosure provides a computer control system programmed to implement the methods of the present disclosure. FIG. 7 illustrates a computer system 701 programmed or otherwise configured to process and/or detect a sample. Computer system 701 can adjust various aspects of the methods and systems of the present disclosure. The computer system may be configured to regulate or communicate with any barrier system or component thereof and/or any processing system or component thereof described herein. For example, the computer system 701 may include or be a controller configured to communicate with fluid flow units, actuators, and/or detectors of the systems described herein.

The computer system 701 includes a central processing unit (CPU, also referred to herein as a "processor" and a "computer processor") 705, which may be a single-core or multi-core processor, or a plurality of processors for parallel processing. Computer system 701 also includes memory or memory location 710 (e.g., random access memory, read only memory, flash memory), electronic storage unit 715 (e.g., a hard disk), communication interface 720 (e.g., a network adapter) for communicating with one or more other systems, and peripheral devices 725 such as cache, other memory, data storage, and/or an electronic display adapter. The memory 710, the storage unit 715, the interface 720, and the peripheral device 725 communicate with the CPU 705 through a communication bus (solid line) such as a motherboard. The storage unit 715 may be a data storage unit (or a data repository) for storing data. The computer system 701 is operably coupled to a computer network ("network") 730 by means of a communication interface 720. The network 730 may be the internet, and/or an extranet, or an intranet and/or an extranet in communication with the internet. In some cases, network 730 is a telecommunications and/or data network. Network 730 may include one or more computer servers that may implement distributed computing, such as cloud computing. In some cases, network 730 may implement a peer-to-peer network with the aid of computer system 701, which may enable devices coupled to computer system 701 to function as clients or servers.

The CPU 705 may execute a series of machine readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as memory 710. Instructions may be directed to the CPU 705, which may then program or otherwise configure the CPU 705 to implement the methods of the present disclosure. Examples of operations performed by the CPU 705 may include fetch, decode, execute, and write back.

CPU 705 may be part of a circuit such as an integrated circuit. One or more other components of system 701 may be included in the circuit. In some cases, the circuit is an Application Specific Integrated Circuit (ASIC).

The storage unit 715 may store files such as drivers, libraries, and saved programs. The storage unit 715 may store user data such as user preferences and user programs. In some cases, computer system 701 may include one or more additional data storage units located external to computer system 701, such as on a remote server in communication with computer system 701 via an intranet or the Internet.

Computer system 701 may communicate with one or more remote computer systems over network 730. For example, computer system 701 may communicate with a user's remote computer system. Examples of remote computer systems include personal computers (e.g., portable PCs), tablet or tablet PCs (e.g., iPad、/>Galaxy Tab), phone, smart phone (e.g.)>iPhone, android enabled device, +.>) Or a personal digital assistant. A user may access computer system 701 via network 730.

The methods described herein may be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of computer system 701, such as memory 710 or electronic storage 715. The machine executable code or machine readable code may be provided in the form of software. During use, the code may be executed by processor 705. In some cases, the code may be retrieved from the storage unit 715 and stored on the memory 710 for quick access by the processor 705. In some cases, electronic storage unit 715 may be eliminated and machine-executable instructions stored on memory 710.

The code may be precompiled and configured for use by a machine having a processor adapted to execute the code, or may be compiled during runtime. The code may be provided in a programming language, which may be selected to enable the code to be executed in a precompiled or just-in-time compiled (as-loaded) manner.

Various aspects of the systems and methods provided herein, such as computer system 701, may be embodied in programming. Aspects of the technology may be considered an "article of manufacture" or "article of manufacture" which generally takes the form of machine (or processor) executable code and/or related data carried or embodied on one type of machine-readable medium. The machine executable code may be stored on an electronic storage unit such as memory (e.g., read only memory, random access memory, flash memory) or a hard disk. A "storage" type medium may include any or all of the tangible memory of a computer, a processor, etc., or related modules thereof, such as various semiconductor memories, tape drives, disk drives, etc., which may provide non-transitory storage for software programming at any time. All or part of the software may sometimes communicate over the internet or various other telecommunications networks. For example, such communication may enable software to be loaded from one computer or processor into another computer or processor, such as from a management server or host into a computer platform of an application server. Accordingly, another type of medium that may carry software elements includes light waves, electric waves, and electromagnetic waves, such as those used across physical interfaces between local devices, through wired and optical landline networks, and various air links. Physical elements carrying such waves, such as wired or wireless links, optical links, etc., may also be considered as media carrying software. As used herein, unless limited to a non-transitory tangible "storage" medium, terms computer or machine "readable medium" and the like refer to any medium that participates in providing instructions to a processor for execution.

Thus, a machine-readable medium, such as a computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium, or a physical transmission medium. Nonvolatile storage media includes, for example, optical or magnetic disks, such as any storage devices in any computer, such as might be used to implement a database as shown in the accompanying drawings. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables, copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier wave transmission media can take the form of electrical or electromagnetic signals, or acoustic or light waves, such as those generated during Radio Frequency (RF) and Infrared (IR) data communications. Thus, common forms of computer-readable media include, for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD, or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, RAM, ROM, PROM and EPROMs, FLASH-EPROMs, any other memory chip or cartridge, a carrier wave transporting data or instructions, a cable or link transporting such a carrier wave, or any other medium from which a computer can read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

The computer system 701 may include an electronic display 735, or be in communication with the electronic display 735, the electronic display 735 including a User Interface (UI) 740 for providing, for example, nucleic acid sequencing information to a user. And the detection result is given to the user. The UI may also present a console for configuring the fluid barrier system of the present disclosure and/or components thereof (e.g., pressure changing devices, environmental units, detectors, immersion shells, movement of detectors, movement of plates, movement of containers, movement of substrates, sample processing, etc.). Examples of UIs include, but are not limited to, graphical User Interfaces (GUIs) and web-based user interfaces.

The methods and systems of the present disclosure may be implemented by one or more algorithms. The algorithm may be implemented in software when executed by the central processing unit 705.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. The invention is not intended to be limited to the specific examples provided in the specification. While the invention has been described with reference to the above description, the description and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it is to be understood that all aspects of the invention are not limited to the specific descriptions, configurations, or relative proportions set forth herein, but are dependent upon various conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. Accordingly, it is contemplated that the present invention also encompasses any such alternatives, modifications, variations, or equivalents. The following claims are intended to define the scope of the invention and their equivalents and methods and structures within the scope of these claims and their equivalents are thereby covered.

The present invention provides embodiments including, but not limited to, the following:

1. a method for processing a biological analyte, comprising:

(a) Providing a barrier between a first region and a second region, wherein the first region comprises a substrate having the biological analyte immobilized adjacent thereto, wherein the barrier maintains the first region under a first atmosphere different from a second atmosphere of the second region; and

(b) Detecting one or more signals from the biological analyte or a change thereof using a detector at least partially contained in the first region while (i) the detector is undergoing motion relative to the substrate, wherein the substrate and the detector are not in direct mechanical contact, and (ii) maintaining the first region under the first atmosphere different from the second atmosphere of the second region.

2. The method of embodiment 1, wherein a portion of the barrier comprises a fluid that moves in its entirety.

3. The method of embodiment 2, wherein the fluid comprises air.

4. The method of any of embodiments 2-3, wherein the portion of the barrier comprises a partial vacuum.

5. The method of any of embodiments 2-4, wherein the portion of the barrier comprises fluid from the first region, the second region, or both.

6. The method of any of embodiments 1-5, wherein the first atmosphere is maintained at a first humidity or first humidity range that is different from a second humidity or second humidity range of the second atmosphere.

7. The method of embodiment 6, wherein the first atmosphere has a relative humidity of greater than 90%.

8. The method of any of embodiments 1-7, wherein the first atmosphere is maintained at a first temperature or within a first temperature range that is different from a second temperature or second temperature range of the second atmosphere.

9. The method of any of embodiments 1-8, wherein the first region comprises a first portion and a second portion, wherein the first portion is maintained under a first partial atmosphere, and wherein the second portion is maintained under a second partial atmosphere different from the first partial atmosphere.

10. The method of embodiment 9, wherein the maintaining the first local atmosphere at a first local temperature or a first local temperature range that is different from a second local temperature or a second local temperature range of the second local atmosphere.

11. The method according to any one of embodiments 9-10, wherein the first local atmosphere is maintained at a first local humidity or a first local humidity range, which is different from a second local humidity or a second local humidity range of the second local atmosphere.

12. The method of any of embodiments 1-11, wherein the detector is an optical detector, and wherein the one or more signals are one or more optical signals or signal changes.

13. The method of any of embodiments 1-12, wherein the barrier comprises a first solid component and a second solid component, wherein the first solid component and the second solid component are not in direct mechanical contact, and wherein the first solid component is movable relative to the second solid component.

14. The method of embodiment 13, wherein a portion of the barrier comprises a bulk moving fluid, and wherein the portion is disposed between the first solid component and the second solid component.

15. The method of any of embodiments 13-14, wherein the detector is fixed relative to the first solid component, and wherein the substrate is fixed in a translational direction relative to the second solid component.

16. The method of any of embodiments 13-15, wherein the substrate is rotatable relative to the second solid component.

17. The method of any of embodiments 13-16, wherein a first portion of the first solid component is disposed between the first region and the second region, and wherein a second portion of the first solid component is disposed between the second region and a third region to form a portion of another barrier configured to maintain the third region under a third atmosphere, the third atmosphere being independent of the first atmosphere and the second atmosphere, wherein a portion of the other barrier comprises a fluid that moves in its entirety, and wherein the third region is movable relative to the first solid component independent of the first region.

18. The method of any one of embodiments 1-17, wherein the second atmosphere is a room atmosphere or an ambient atmosphere.

19. The method of any of embodiments 1-18, wherein a first portion of the detector is in the first region and a second portion of the detector is in the second region.

20. The method of embodiment 19, wherein the first portion of the detector comprises an optical imaging objective at least partially immersed in immersion fluid in contact with the substrate in the first region.

21. The method of any one of embodiments 1-20, wherein the biological analyte is a nucleic acid molecule, and further comprising identifying the sequence of the nucleic acid molecule or derivative thereof based at least in part on the one or more signals or changes thereof.

22. The method of any of embodiments 1-21, wherein the movement comprises one or more selected from the group consisting of: (i) Substantially linear motion relative to the substrate and (ii) substantially non-linear motion.

23. The method of any of embodiments 1-22, wherein the detector performs a rotational motion relative to the substrate.

24. The method of any of embodiments 1-22, wherein the detector performs translational movement relative to the substrate.

25. The method of any of embodiments 1-22, wherein the detector performs translational and rotational movement relative to the substrate.

26. The method of any of embodiments 1-25, wherein in (b) the detector scans the substrate along a substantially linear scan path.

27. The method of any of embodiments 1-25, wherein in (b) the detector scans the substrate along a substantially non-linear scan path.

28. The method of embodiment 27, wherein in (b) the detector scans the substrate along one or more scan paths selected from the group consisting of rings, spirals, and arcs.

29. A method for processing a biological analyte, comprising:

(a) Providing a barrier between a first region and a second region, wherein the first region comprises the biological analyte, wherein the barrier maintains the first region under a first atmosphere different from a second atmosphere of the second region, wherein a portion of the barrier comprises a bulk moving fluid; and

(b) One or more signals from the biological analyte or a change thereof are detected using a detector at least partially contained in the first region while maintaining the first region under the first atmosphere different from the second atmosphere of the second region.

30. The method of embodiment 29, wherein the portion of the barrier comprises fluid from the first region, the second region, or both.

31. The method of any of embodiments 29-30, wherein the first atmosphere is maintained at a first humidity or first humidity range that is different from a second humidity or second humidity range of the second atmosphere.

32. The method of embodiment 31, wherein the first atmosphere has a relative humidity of greater than 90%.

33. The method of any of embodiments 29-32, wherein the first atmosphere is maintained at a first temperature or within a first temperature range that is different from a second temperature or second temperature range of the second atmosphere.

34. The method of any of embodiments 29-33, wherein the first region comprises a first portion and a second portion, wherein the first portion is maintained under a first partial atmosphere, and wherein the second portion is maintained under a second partial atmosphere different from the first partial atmosphere.

35. The method of embodiment 34, wherein the maintaining the first local atmosphere at or within a first local temperature or a first local temperature range that is different from a second local temperature or a second local temperature range of the second local atmosphere.

36. The method of any of embodiments 34-35, wherein the first local atmosphere is maintained at a first local humidity or first local humidity range that is different from a second local humidity or second local humidity range of the second local atmosphere.

37. The method of any one of embodiments 29-36, wherein (b) comprises moving the detector relative to the biological analyte upon detection.

38. The method of any of embodiments 29-37, wherein the detector is an optical detector, and wherein the one or more signals or changes thereof are one or more optical signals or changes thereof.

39. The method of any of embodiments 29-38, wherein the barrier comprises a first solid component and a second solid component, wherein the first solid component and the second solid component are not in mechanical contact, and wherein the first solid component is movable relative to the second solid component.

40. The method of embodiment 39, wherein the portion of the barrier comprising the fluid is disposed between the first solid component and the second solid component.

41. The method of any one of embodiments 39-40, wherein the detector is immobilized relative to the first solid component, and wherein the biological analyte is immobilized in a translational direction relative to the second solid component.

42. The method of any of embodiments 39-41, wherein a first portion of the first solid component is disposed between the first region and the second region, and wherein a second portion of the first solid component is disposed between the second region and a third region to form a portion of another barrier configured to maintain the third region under a third atmosphere independent of the first atmosphere and the second atmosphere, wherein a portion of the other barrier comprises a fluid, and wherein the third region is movable relative to the first solid component independent of the first region.

43. The method of any of embodiments 29-42, wherein the second atmosphere is a room atmosphere or an ambient atmosphere.

44. The method of any of embodiments 29-43, wherein a first portion of the detector is in the first region and a second portion of the detector is in the second region.

45. The method of embodiment 44, wherein the first portion of the detector comprises an optical imaging objective at least partially immersed in an immersion fluid in contact with the biological analyte in the first region.

46. The method of any one of embodiments 29-45, wherein the biological analyte is a nucleic acid molecule, and further comprising identifying the sequence of the nucleic acid molecule or derivative thereof based at least in part on the one or more signals or signal changes.

47. The method of any of embodiments 29-46, wherein the fluid comprises air.

48. A system for processing an analyte, comprising:

a first region configured to comprise (i) a substrate comprising the analyte immobilized thereabout, and (ii) at least a portion of a detector; and

a barrier disposed between the first region and the second region, wherein the barrier is configured to maintain the first region at a first atmosphere when the detector and the substrate are in relative motion with respect to each other to detect one or more signals from the analyte or a change thereof, the first atmosphere being different from a second atmosphere of the second region.

49. The system of embodiment 48, wherein a portion of the barrier is configured to include a fluid in bulk motion.

50. The system of embodiment 49, wherein the portion of the barrier is configured to be under vacuum.

51. The system of any of embodiments 49-50, wherein the portion of the barrier is configured to include fluid from the first region, the second region, or both the first region and the second region.

52. The system of any of embodiments 48-51, wherein a portion of the barrier is configured to include air.

53. The system of any of embodiments 48-52, wherein the barrier is configured to maintain the first region at a first humidity or within a first humidity range, wherein the first humidity or first humidity range is different from a second humidity or second humidity range of the second region.

54. The system of embodiment 53, wherein the first atmosphere has a relative humidity of greater than 90%.

55. The system of any of embodiments 48-54, wherein the barrier is configured to maintain the first region at a first temperature or within a first temperature range, wherein the first temperature or first temperature range is different than a second temperature or second temperature range of the second region.

56. The system of any of embodiments 48-55, wherein the first region comprises a first portion and a second portion, wherein the barrier is configured to maintain the first portion under a first local atmosphere and to maintain the second portion under a second local atmosphere different from the first local atmosphere.

57. The system of embodiment 56, wherein the barrier is configured to maintain the first local atmosphere at a first local temperature or within a first local temperature range that is different from a second local temperature or second local temperature range of the second local atmosphere.

58. The system of any of embodiments 56-57, wherein said barrier is configured to maintain said first local atmosphere at or within a first local humidity or first local humidity range that is different from a second local humidity or second local humidity range of said second local atmosphere.

59. The system of any of embodiments 48-58, wherein the detector is at least partially contained in the first region.

60. The system of embodiment 59, wherein the detector is an optical detector, and wherein the one or more signals are one or more optical signals or signal variations.

61. The system of any of embodiments 59-60, wherein a first portion of the detector is in the first region and a second portion of the detector is in the second region.

62. The system of any of embodiments 59-61, wherein the first portion of the detector comprises an optical imaging objective configured to be at least partially immersed in an immersion fluid in contact with the substrate when the substrate is in the first region.

63. The system of any of embodiments 59-62, wherein the detector is configured to move when the substrate is stationary.

64. The system of any of embodiments 59-63, wherein the substrate is configured to move when the detector is stationary.

65. The system of any of embodiments 48-64, wherein the barrier comprises a first solid component and a second solid component, wherein the first solid component and the second solid component are not in direct mechanical contact with each other, and wherein the first solid component and the second solid component are movable relative to each other.

66. The system of embodiment 65, wherein a portion of the barrier is configured to include a fluid in bulk motion, and wherein the portion is disposed between the first solid component and the second solid component.

67. The system of any of embodiments 65-66, wherein the detector is configured to be fixed relative to the first solid component, and wherein the substrate is configured to be fixed relative to the second solid component.

68. The system of any of embodiments 65-66, wherein the detector is configured to be fixed relative to the first solid component, and wherein the substrate is configured to be rotatable relative to the second solid component.

69. The system of any of embodiments 48-68, wherein a first portion of the first solid component is disposed between the first region and the second region, and wherein a second portion of the first solid component is disposed between the second region and a third region to form a portion of another barrier configured to maintain the third region under a third atmosphere, the third atmosphere being independent of the first atmosphere and the second atmosphere, wherein a portion of the other barrier comprises a fluid that moves in its entirety, and wherein the third region is movable relative to the first solid component independent of the first region.

70. The system of any of embodiments 48-69, wherein the second atmosphere is a room atmosphere or an ambient atmosphere.

71. A system for processing or analyzing an analyte, comprising:

a chamber and a lid, wherein the chamber comprises a first region configured to contain (1) a substrate containing the analyte immobilized thereabout, and (2) at least a portion of a detection unit, and wherein the lid is configured to be disposed adjacent to the chamber; and

a fluid flow unit configured to provide a fluid in an overall motion at a location disposed between the chamber and the lid when the lid is disposed adjacent the chamber such that the first region is maintained at a first atmosphere that is different from a second atmosphere of a second region outside the first region.

72. The system of embodiment 71, wherein the integrally-moving fluid is configured to provide a partial vacuum between the chamber and the lid.

73. The system of any of embodiments 71-72, wherein the fluid flow unit is configured to provide the integrally-moving fluid using fluid from the first region, the second region, or both.

74. The system of any of embodiments 71-73, wherein the fluid comprises air.

75. The system of any of embodiments 71-74, wherein the fluid flow unit is configured to maintain the first region at a first humidity or a first range of humidity, wherein the first humidity or first range of humidity is different from a second humidity or second range of humidity of the second region.

76. The system of embodiment 75, wherein the first atmosphere has a relative humidity of greater than 90%.

77. The system of any of embodiments 71-76, wherein the fluid flow unit is configured to maintain the first region at a first temperature or a first temperature range, wherein the first temperature or first temperature range is different than a second temperature or second temperature range of the second region.

78. The system of any of embodiments 71-77, wherein the first region comprises a first portion and a second portion, wherein the fluid flow unit is configured to maintain the first portion under a first partial atmosphere and to maintain the second portion under a second partial atmosphere different from the first partial atmosphere.

79. The system of embodiment 78, wherein the fluid flow unit is configured to maintain the first local atmosphere at a first local temperature or within a first local temperature range that is different from a second local temperature or second local temperature range of the second local atmosphere.

80. The system of any of embodiments 78-79, wherein the fluid flow unit is configured to maintain the first local atmosphere at a first local humidity or within a first local humidity range that is different from a second local humidity or second local humidity range of the second local atmosphere.

81. The system of any one of embodiments 71-80, wherein said detection unit is at least partially contained in said first region.

82. The system of embodiment 81, wherein the detection unit is an optical detection unit.

83. The system of any of embodiments 81-82, wherein a first portion of the detection unit is in the first region and a second portion of the detection unit is in the second region.

84. The system of any of embodiments 81-83, wherein the first portion of the detection unit comprises an optical imaging objective configured to be at least partially immersed in an immersion fluid in contact with the substrate in the first region.

85. The system of any of embodiments 81-84, wherein the detection unit is configured to move while the substrate is stationary.

86. The system of any of embodiments 81-85, wherein the substrate is configured to move when the detection unit is stationary.

87. The system of any of embodiments 81-86, wherein the relative motion comprises one or more selected from the group consisting of: (i) Substantially linear motion and (ii) substantially non-linear motion.

88. The system of any of embodiments 81-87, wherein the detection unit is configured to be fixed relative to the cover.

89. The system of any of embodiments 81-88, wherein the substrate is configured to be rotatable relative to the chamber.

90. The system of any of embodiments 71-89, wherein the detection unit comprises one or more optics.

91. The system of any of embodiments 71-90, wherein the detection unit comprises a sensor configured to capture a signal from the analyte.

92. The system of any of embodiments 71-91, wherein the chamber is not in mechanical contact with the cap.

93. The system of any of embodiments 71-92, wherein the cover is configured to move relative to the chamber, or vice versa.

94. The system of any of embodiments 71-93, wherein the fluid flow unit is configured to: to maintain the first region under the first atmosphere while the detection unit and the substrate are moving relative to each other.

95. The system of any of embodiments 71-94, wherein the fluid flow unit is configured to: a negative pressure is generated in the location disposed between the chamber and the cover.

96. The system of any of embodiments 71-95, wherein a first portion of the cover is disposed between the first region and the second region, and wherein a second portion of the cover is disposed between the second region and a third region, wherein a second fluid flow unit is configured to provide fluid for unitary movement to maintain the third region under a third atmosphere, the third atmosphere being independent of the first atmosphere and the second atmosphere, and wherein the third region is movable relative to the cover independent of the first region.

97. The system of any of embodiments 71-96, wherein the second atmosphere is a room atmosphere or an ambient atmosphere.

98. The system of any of embodiments 71-97, further comprising a controller operably coupled to the fluid flow unit, wherein the controller is configured to direct the fluid flow unit to cause the fluid to perform the overall motion.

99. A system, comprising:

an imaging objective configured to detect a signal or signal change from an analyte coupled to a substrate;

a housing configured to hold a volume of fluid between the imaging objective and the substrate;

a fluid source configured to comprise an aqueous solution; and

a fluid flow unit configured to deliver the volume of fluid from the fluid source to the housing.

100. The system of embodiment 99, wherein the aqueous solution comprises a wash solution.

101. The system of any of embodiments 99-100, wherein the aqueous solution comprises an immersion buffer solution comprising a salt, a surfactant, and a buffer.

102. The system of any of embodiments 99-101, wherein the aqueous solution has a pH of 8.0 to 9.0.

103. The system of any of embodiments 99-102, further comprising the substrate.

104. The system of embodiment 103, wherein the substrate comprises a fluid layer comprising a second aqueous solution.

105. The system of embodiment 104, wherein the aqueous solution and the second aqueous solution comprise different compositions.

106. The system of embodiment 104, wherein the aqueous solution and the second aqueous solution comprise the same composition.

107. A method, comprising:

(a) Contacting an imaging objective with a substrate fluid through a volume of fluid, wherein the fluid comprises a first aqueous solution, wherein the substrate comprises (i) an analyte immobilized near the substrate, and (ii) a fluid layer adjacent to the substrate, wherein the fluid layer comprises a second aqueous solution; and

(b) Imaging the analyte through the volume of fluid with the imaging objective.

108. The method of embodiment 107, further comprising moving the imaging objective relative to the substrate while maintaining fluid contact between the imaging objective and the substrate.

109. The method of any of embodiments 107-108, further comprising moving the substrate relative to the imaging objective while maintaining fluid contact between the imaging objective and the substrate.

110. The method of any of embodiments 107-109, wherein the volume of fluid has a thickness of about 200 micrometers (μιη) to 500 μιη.

111. The method of any of embodiments 107-110, wherein the fluid layer has a thickness of about 5 μιη to 50 μιη.

112. The method of any of embodiments 107-111, further comprising (i) breaking fluid contact between the imaging objective and the substrate, and (ii) bringing the imaging objective and the substrate into second fluid contact.

113. The method of embodiment 112, wherein after (i) at least a portion of the volume of fluid remains in fluid contact with the imaging objective.

114. The method of any of embodiments 112-113, wherein after (i), at least a portion of the volume of fluid remains in contact with the substrate fluid.

115. The method of any of embodiments 107-114, wherein the first aqueous solution comprises a wash solution.

116. The method of any of embodiments 107-115, wherein the first aqueous solution comprises an immersion buffer solution comprising a salt, a surfactant, and a buffer.

117. The method of any of embodiments 107-116, wherein the first aqueous solution has a pH of 8.0 to 9.0.

118. The method of any of embodiments 107-117, wherein the first aqueous solution and the second aqueous solution comprise different compositions.

119. The method of any of embodiments 107-117, wherein the first aqueous solution and the second aqueous solution comprise the same composition.

120. A method, comprising:

(a) Contacting the imaging objective with an analyte immobilized adjacent to a substrate through a volume of fluid, wherein the substrate comprises a fluid layer comprising a second aqueous solution; and

(b) Imaging the analyte through the volume of fluid with the imaging objective.

121. The method of embodiment 120, further comprising moving the imaging objective relative to the analyte while maintaining fluid contact between the imaging objective and the analyte.

122. The method of any of embodiments 120-121, further comprising moving the analyte relative to the imaging objective while maintaining fluid contact between the imaging objective and the analyte.

123. The method of any of embodiments 120-122, wherein the volume of fluid has a thickness of about 200 μιη to 500 μιη.

124. The method of any of embodiments 120-123, wherein the fluid layer has a thickness of about 5 μιη to 50 μιη.

125. The method of any of embodiments 120-124, further comprising (i) breaking fluid contact between the imaging objective and the analyte, and (ii) bringing the imaging objective and the analyte into fluid contact a second time.

126. The method of embodiment 125, wherein after (i) at least a portion of the volume of fluid remains in fluid contact with the imaging objective.

127. The method of any of embodiments 125-126, wherein after (i), at least a portion of the volume of fluid remains in contact with the analyte fluid.

128. The method of any of embodiments 120-127, wherein the first aqueous solution comprises a wash solution.

129. The method of any of embodiments 120-128, wherein the first aqueous solution comprises an immersion buffer solution comprising a salt, a surfactant, and a buffer.

130. The method of any of embodiments 120-129, wherein the first aqueous solution has a pH of 8.0 to 9.0.

131. The method of any of embodiments 120-130, wherein the first aqueous solution and the second aqueous solution comprise different compositions.

132. The method of any of embodiments 120-130, wherein the first aqueous solution and the second aqueous solution comprise the same composition.

133. A system for processing or analyzing an analyte, comprising:

a chamber and a lid, wherein the chamber comprises an interior region and is configured to include a substrate configured to immobilize the analyte in proximity thereto, wherein the lid is configured to be disposed adjacent to the chamber; and

an environmental unit configured to maintain a first local environment, a second local environment, and a third local environment within the interior region, wherein the environmental unit is configured to (i) maintain the first local environment within a first temperature or temperature range, (ii) maintain the second local environment within a second temperature or temperature range, and (iii) maintain the third local environment within a third temperature or temperature range,

wherein the first local environment is disposed above the second local environment and the third local environment, and wherein the first local environment is located at or near the lid, and

Wherein the second local environment is disposed at or near a surface of the substrate,

wherein the third local environment is disposed under the first local environment and the second local environment, and

wherein the first temperature or temperature range is higher than the second temperature or temperature range and the third temperature or temperature range, and wherein the second temperature or temperature range is lower than the third temperature or temperature range.

Examples

Example 1 imaging for nucleic acid molecule sequencing

Fig. 8 shows an example of an image generated by imaging a substrate immobilized with a biological analyte in a sample environment of a barrier system of the present disclosure. A substrate 810 comprising a substantially planar array has immobilized thereon a plurality of biological analytes, such as nucleic acid molecules, at a plurality of individually addressable locations 820. The individually addressable locations may be arranged randomly or in an ordered pattern. The biological analyte may be attached to a bead, which is immobilized on an array. A single bead may contain multiple analytes. The beads may be associated with individually addressable locations. A plurality of fluorescent probes (e.g., a plurality of fluorescently labeled A, T, C or G containing nucleotides or the like) are dispensed onto the substrate 810 with the aid of one or more handling units (e.g., reagent dispensers) of a chemical processing station. In some embodiments, the substrate is configured to rotate relative to the shaft. The substrate 810 is then subjected to conditions sufficient to perform a reaction between at least one probe of the plurality of probes and the biological analyte to couple the at least one probe to the biological analyte. With the aid of one or more operating units, the uncoupled probe is flushed away. At the detection station, while maintaining the fluid barrier, coupling of at least one probe to the biological analyte is detected using a photometric method that includes imaging (e.g., by scanning or fixed field imaging) at least a portion of the substrate 810 and measuring the signal of each individually addressable location 820. Nucleic acid molecules comprising nucleotides complementary to fluorescent probes are fluorescent in individually addressable locations 820. The operations may then be iterated at the respective stations and the signals from the images are compared with the signals from the previous images of the same substrate to generate a trace of the signals in time for each biological analyte in each individually addressable location 820. The sequence of the plurality of fluorescent probes is known for each iteration of the operation, thereby generating a known sequence of analytes in each of the individually addressable locations 820.

Example 2 Signal processing

Fig. 9 shows signal data processed by imaging a substrate immobilized with a biological analyte in a sample environment of a barrier system of the present disclosure. The substrate comprising a substantially planar array has immobilized thereon nucleic acid molecules from E.coli (E.coli). Sequencing by synthesis was performed using flow-based chemistry using the processing system described herein. Imaging is performed while maintaining the fluid barrier of the barrier system, as described elsewhere herein. Panel (a) of fig. 9 shows the signal distribution for a set of hundreds of colonies, each of which is a replica of a single synthetic single template. The x-axis is labeled with the length of the sequence after each cycle (e.g., each chemical flow operation). In panel (B) of fig. 9, the same data has been processed using a parametric model. The parametric model takes into account the different template counts (amplitudes) and background levels for each colony. The signal is deconvolved using lead and lag phase models, and also taking into account the global signal loss per cycle. In the embodiment described herein, the nominal phase is 0.54% lag, 0.41% lead, and 0.45% signal loss. Residual systematic variations may be due to signal variations in sequence context that may be further corrected using other algorithms (not shown).

Claims

1. A method for processing a biological analyte, comprising:

2. The method of claim 1, wherein a portion of the barrier comprises a fluid that moves in bulk.

3. The method of claim 2, wherein the fluid comprises air.

4. A method for processing a biological analyte, comprising:

5. A system for processing an analyte, comprising:

6. A system for processing or analyzing an analyte, comprising:

7. A system, comprising:

a fluid source configured to comprise an aqueous solution; and

8. A method, comprising:

(b) Imaging the analyte through the volume of fluid with the imaging objective.

9. A method, comprising:

(b) Imaging the analyte through the volume of fluid with the imaging objective.

10. A system for processing or analyzing an analyte, comprising: